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Friedlaqnders Use Of The Microscope. 




THE USB 



MICROSCOPE 



IN 



CLINICAL AND PATHOLOGICAL 
EXAMINATIONS. 



/ BY 

De. carl friedlaender, 

PRIVAT-DOCENT IN PATHOLOGICAL ANATOMY AT BERLIN. 



SECOND EDITION, ENLAEGED AND IMPEOVED, WITH A CHEOMO-LITHOGEAPH. 



TRANSLATED, WITH THE PERMISSION OF THE AUTHOR, 
By HENRY C. COE, 

M. D., M. R. C. S., L. R. C. P. (LONDON), 
PATHOLOGIST TO THE WOMAN' S HOSPITAL IN THE STATE OF NEW YORK. 



'/?lo2 & 



NEW YORK: 
D. APPLETON AND COMPANY, 

1, 3, and 5 BOND STREET. 

1885. 



^ 



■f 



Copyright, 1885, 
By D. APFLETON AND COMPANY. 



PREFACE TO THE FIRST GERMAN EDITION. 



The author has been frequently requested to give 
a brief description of those methods which are adopted 
in microscopical examinations, conducted for diagnos- 
tic and pathological purposes. These same methods, 
which twenty years ago were extremely simple, have 
gradually become more complicated and have, in 
many respects, been very essentially improved and 
refined. A large number of the surprising advances 
which have been made of late years in the field of 
vegetable parasites, are the direct results of improve- 
ments in technique. 

If, therefore, every one who aspires to follow the 
progress of pathology must become acquainted with 
recent methods, this is still more incumbent upon him 
who desires to undertake microscopical examinations 
for clinical and pathological ends. A concise state- 
ment of the processes employed in pathological his- 
tology has hitherto been wanting, and its absence has 
been deeply felt by many ; this book aims at supply- 
ing the deficiency. The fact that examinations for 
schizomycetes have been treated quite exhaustively, 
and have received especial preference, ought to meet 



iv PREFACE TO THE FIRST GERMAN EDITION. 

with universal approval. It has frequently been 
necessary to enter rather deeply into the diagnostic 
and prognostic significance of discoveries ; particular 
stress was laid upon this in discussing the subject of 
tubercle-bacilli in sputa, and in deciding between 
erosion and carcinoma of the uterus. 

May this little volume serve as a guide to the 
beginner in this study, which is as fascinating as it is 
difficult. Perhaps the expert, too, may find here and 
there a useful hint. 

Carl Friedlaekder. 

City Hospital, Beelin, August, 1882. 



PREFACE TO THE SECOND GERMAN EDITION. 



A second edition of this little book has become 
necessary after the lapse of a comparatively short 
period. I have aimed to select from the many new 
contributions on technology, and to add to the text, 
those which present essential improvements as re- 
gards my purposes. I have also, in accordance with 
the wishes of many, presented in the appended col- 
ored plate a comparison of the most important and 
characteristic pathogenic schizomycetes. I am in- 
debted for the drawings to my honored friend and 
fellow-worker Dr. Gram, of Copenhagen. They have 
all been executed on the same scale — one to one 
thousand. 

The Author. 

Beklist, April, 1884. 



CONTENTS. 



CHAPTER I. 

THE MICROSCOPE. 



PAGE 

1. The stand. Abbess apparatus . 1 

2. Objectives. Water- and oil-immersion lenses .... 4 

3. Eye-pieces. Accessory apparatus. Combinations 7 

CHAPTER II. 

ACCESSOEIES. 

1. Illuminating lamp 8 

2. Glass apparatus 9 

3. Metallic instruments 10 

4. The microtome. Thick and thin sections ; further treatment of 

sections 11 

CHAPTER III. 

REAGENTS. MICRO-CHEMISTRY. 

Artificial products 19 

Micro- chemical examinations 21 

1. Distilled water 22 

2. Salt-solution of 0*8 per cent. Indifferent fluid . . . .23 

3. Absolute alcohol. Hardening 24 

4. Ether and chloroform. The removal of fat . . . . 27 

5. Acids 28 

a. Sulphuric, hydrochloric, and nitric acids. Decalcification . 28 

b. Acetic acid 30 

c. Picric acid 33 

d. Chromic acid ; chromates. Muller's fluid . . . .34 

6. Alkalies. Liquor potassaB and liquor sodse. Ammonia . . 36 



viii CONTENTS. 

PAGE 

7. Glycerin ... 38 

8. Acetate of potassium . .40 

9. Oil of doves, Canada balsam 40 

Keagents used in staining. The principles of staining . . .42 

10. Iodine 46 

Glycogen 47 

Corpora amylacea 48 

Amyloid material . . . . . . . . . .48 

11. Carmine . . . . . . . . . . .51 

a. Ammonia-carmine 51 

~b> Picro-carmine 54 

c. Borax-carmine 56 

d. Alum-carmine . . .56 

e. Cochineal-alum solution 57 

f. Lithium- carmine 57 

12. Hematoxylin 58 

Weigert's method of staining nerve-fibers 58 

13. Eosin 60 

14. Aniline-black (nigrosin). Aniline-blue 62 

Staining of nuclei . 62 

Food-cells 66 

Staining of amyloid substance QQ 

15. Identification and staining of schizomycetes . . . .67 
a. Identification of schizomycetes when unstained . . .68 
t. Staining of micrococci 71 

c. Gram's method 75 

d. Staining of tubercle-bacilli 78 

16. The noble metals. 

a. Silver 86 

b. Gold 88 

c. Osmic acid . . . 89 

17. Ammonium sulphide 90 

CHAPTER IV. 

OTHEE METHODS OF PEEPAEATIOK. 

1. Boiling 93 

2. Drying 94 

3. Artificial digestion 94 

4. Imbedding. Celloidine 97 

5. The process of injection 100 

a. Injecting materials . . 100 

J). Injecting apparatus . 103 

6. Preservation of specimens 106 



CONTENTS. i x 

CHAPTER V. 

THE OBSERVATION OF LIVING TISSUES. 

PAGE 

The circulation. Inflammation 109 

1. The frog's web 110 

2. The tongue 110 

3. The mesentery Ill 

4. The cornea 112 

CHAPTER VI. 

THE EXAMINATION OF FLUIDS. 

Vital properties of the suspended elements. Amoeboid movements 114 

The form of the elements 116 

Examination of tissue-fluid 117 

Examination of micro-organisms 118 

Koch's method of staining dried preparations 121 

1. Blood 126 

a. Diminution of the number of red blood-corpuscles in anaemia 127 

b. Change in the size and shape of the red blood-corpuscles. 

Nucleated red blood-corpuscles 128 

c. Increase in the number of white blood-corpuscles. Leucocy- 

tosis and leucaemia. Changes in the granular protoplasm . 129 

d. Other cell-elements in the blood. Worms and schizomycetes 131 

e. The examination of blood-stains. Haemin-crystals. Haema- 

toidin 134 

2. Sputa . . .136 

a. Oral fluids 137 

5. Products of the respiratory mucous membrane . . . 141 

c. Elastic fibers. Fibrinous exudations. Asthma-crystals . 145 

d. Schizomycetes. Tubercle-bacilli. Pneumococci . . . 146 

3. Pus. 

a. Pus-corpuscles and fatty degenerated cells .... 155 

l. Foreign substances 156 

c. Schizomycetes and actinomycetes 158 

4. Urine. 

a. Precipitates and crystals ...'.... 159 

b. Casts 161 

c. Pus- and mucus-corpuscles. Epithelial cells . . . .163 

d. Tumor-elements 164 

e. Entozoa 164 

/. Vegetable parasites 164 



x CONTENTS. 

5. Secretions of the genital tract. paos 

a. The vaginal secretion 166 

l. Fluids from the uterus 167 

Carcinoma or erosion 169 

c. Gonorrheal secretion 173 

d. Semen and the prostatic secretion 174 

6. Contents of the stomach and intestine. 

a. Eemains of food 175 

5. Epithelial cells, mucus, etc 176 

c. Entozoa 177 

d. Vegetable parasites . 177 

7. Exudations. Contents of cysts . . . . . .179 

CHAPTER VII. 

The examination of solid elements of the body, extirpated tumors, 

etc. . 183 



THE 



USE OF THE MICROSCOPE. 



THE MICROSCOPE. 

In regard to the choice of a microscope, the prin- 
ciple is to be established that the lenses and the stage 
must be free from defects. Do not be led to purchase 
an instrument of inferior quality, because the price is 
a little lower ; most of the objects which we have to 
examine with the microscope present so many diffi- 
culties, their outlines are so delicate, etc., that it is 
only under the most favorable circumstances that we 
succeed in bringing them into view perfectly, and 
without great loss of time. Therefore, it is best to 
select at the outset an instrument of a well-known 
and reliable make. If one of the lenses, or other parts, 
which have been furnished, does not satisfy all of the 
proper requirements, send them back at once, for 
nothing is more unpleasant than a year's struggle 
with an imperfect microscope. 

On the contrary, it is by no means necessary to pur- 
chase the strongest objectives at the start, since they 
materially increase the price of instruments. It is 
better for the beginner to use only low and mod- 
erate powers at first — three hundred at the highest ; 



2 THE USE OF THE MICROSCOPE. 

the management of stronger lenses is beset with so 
many difficulties, and demands such extreme accuracy, 
that it is decidedly advisable to prepare one's self for 
this by previously working for some time with lower 
powers. 

1. The Stajstd (Abbe's Apparatus). — The stand 
must be so arranged that it can be used even with 
the strongest objectives ; above all, the action of the 
micrometer-screw must be sufficiently delicate. The 
stage must be large, firm, and steady, and the opening 
not too .small, so that a section of the spinal cord, for 
example, can be examined in toto, under a low power 
and with the diaphragm removed. The contrivance 
for revolving the stand is, as a rule, superfluous. 

The cylindrical diaphragms (the disk-diaphragms 
are less perfect) must, as a matter of course, be 
exactly centered, and readily exchangeable. Narrow 
diaphragms are employed in the case of unstained 
objects, in order to bring the structures sharply into 
view when higher powers are used ; with low powers 
it is usually necessary to attach a wider diaphragm, 
so that the entire field of vision may be utilized. A 
condenser, or Abbe's apparatus, is desirable in all 
cases, and it is necessary in examinations of schizo- 
mycetes. The rays of light, reflected from the mir- 
ror upon the lens of the condenser, are so refracted 
by this that they meet in a single point (focus), and 
this point lies exactly at the position of the object. 
In this way the object receives an immense quantity 
of light, not only a collection of nearly parallel rays 
from below, as in the ordinary examination with a 
narrow diaphragm, but an entire cone, with the larg- 



THE MICROSCOPE. 3 

est possible angle of divergence, at the apex of which 
lies the object. For this reason the delicate contours 
of the transparent object, so far as they depend upon 
differences of refraction, are almost entirely lost ; as 
Koch expresses it, the contours are " extinguished." 
The stained portions of the specimen, which would 
otherwise have been partly or wholly concealed by 
the outlines of the unstained portions, appear so 
much the more distinct. Koch terms this "isola- 
tion of the stained image." Thus, by the examination 
with the open condenser, we very often succeed in 
recognizing as such deeply-stained micro-organisms, 
or other small colored bodies, which under the ordi- 
nary illumination are concealed by the image of the 
structure, and thus are only indistinct or even quite 
invisible. The angle of divergence of the light-cone 
amounts, when Abbe's condenser is used, to 120° ; the 
condensers previously manufactured usually give a 
much smaller angle, and are consequently insufficient. 
Beneath the lens of the condenser is a disk, provided 
with diaphragms of different sizes which are inter- 
changeable ; by employing a narrow diaphragm you 
naturally obtain an illumination quite similar to that 
furnished by a narrow cylindrical one. In order to 
isolate the colored image, the diaphragms are removed 
entirely. The other complicated arrangements of 
Abbe's apparatus have not, up to the present time, 
been essential to my purposes. On the contrary, the 
use of illumination by means of the open condenser 
is of very great value for all stained preparations, 
while in many difficult examinations it is even indis- 
pensable. We have to thank Koch for the introduc- 



4 THE USE OF THE MICROSCOPE. 

tion of this method.* In choosing a stand, care 
must be taken that a well-made condenser, with a 
large focal angle, or an Abbe's apparatus, is, or at 
least can be, attached. 

2. Objectives. Water akd Oil Immersions. — 
As regards the choice of the lenses, the necessary ob- 
jectives are : 

(1.) Quite a weak one, having a focal distance of 
about thirty millimetres, which with the medium 
ocular gives a magnifying power of about twenty ; 
this is for the general inspection of large sections, 
those, for example, from the brain and spinal cord, 
liver, and kidney, for the observation of trichinae, etc. 

(2.) A moderately weak lens, with a focal distance 
of about fifteen millimetres, such as will attain a 
magnifying power of about sixty or eighty. 

(3.) A moderately strong one, with a focal distance 
of four millimetres, giving a power of three hundred. 

(4.) A rather strong immersion-lens, having a fo- 
cal distance of one and one half or two millimetres, 
for more delicate examinations. 

The powers of eighty and three hundred will be 
most useful for our purposes. 

The immersion systems, f which are employed in 
order to obtain very strong magnifying powers, de- 
mand for their management a certain degree of care 
and experience. As already stated, it is best for the 

* R. Koch. " Untersuchungen fur Aetiologie der Wundinfections- 
krankheiten." Leipzig, 1878. 

t The usual designation of the immersion systems, as T V, xg-, refers 
to their equivalent focal distance, which, according to the English cus- 
tom, is expressed in inches: ■£% corresponds to 2 millimetres, -^ to 1*3 
millimetres. 



THE MICROSCOPE. 5 

beginner not to use them at first. It is impossible, 
without entering into rather exact physical demon- 
strations, to explain the advantages of immersion. 
This will be enough for us : as the rays pass from 
the upper surface of the cover-glass into the air, and 
again from the air into the under surface of the ob- 
jective-lens of the microscope, those rays alone remain 
unchanged which impinge upon these surfaces ver- 
tically ; the oblique rays are changed in their course, 
and the more acute the angle at which they fall, the 
more they are refracted. 

Now, if we designate as the angle of divergence 
of an objective that angle which is formed by the 
diverging rays proceeding from any point in the 
object to the extreme edge of the lens (which are 
reunited at a point in the image above the objec- 
tive), then it is evident that this angle of divergence 
can not be increased above a certain degree with dry 
objectives, if the clearness of the image is to be pre- 
served.* For the peripheral rays, which, because of 
their oblique passage from the glass to the air, and 
from the air to the glass, are twice deflected, appear to 
proceed from a different point than the more central 
rays ; in addition to the spherical aberration of light, 
there is another element which prevents the employ- 
ment of an angle of divergence as large as possible. 
This evil is essentially lessened if we interpose a layer 
of water between the cover-glass and lens (water-im- 
mersion), since the difference in refractive power be- 
tween water and glass is much less than that between 

* This disadvantage is avoided, at least in part, by the so-called 
" correction." Compare the following note. 



6 THE USE OF TEE MICROSCOPE, 

air and glass; it can be almost entirely removed, 
however, if a fluid is interposed which has the 
same refractive power as glass (homogeneous or oil- 
immersion). Oil of cedar, or a mixture of fennel and 
castor oils, is used for this purpose ; * a mixture of 
chloral hydrate and glycerin has been recently recom- 
mended. These oil-immersion lenses are the master- 
pieces of our opticians ; Abbe, Zeiss, and Stephen- 
son have acquired a great deal of credit by their in- 
troduction. 

With the increase in the angle of divergence, not 
only is the clearness of the image augmented, but 
the power of differentiation — the so-called resolving 
power — of the instrument is increased to a degree 
not hitherto attainable. 

The use of oil-immersions is not unattended with 
difficulties, even for a skilled microscopist, who is 
accustomed to neat and delicate work. 

In the case of water-immersion lenses, one veiy 
soon learns to determine the size of the drop, which 
is placed, by means of a glass rod, upon the front 
lens of the system, or upon the glass which covers 
the object ; further than this, one has only to prevent 

* This is only one of the advantages offered by the immersion sys- 
tems ; besides this they have, by reason of the relations of reflection and 
refraction, more light than dry systems of the same focal angle, as can 
readily be demonstrated. In order to correct the influence of cover- 
glasses of different thicknesses, the powerful dry and water immersion 
objectives are constructed in so-called " correction-mountings,'- which 
allow the lenses of which the system is composed to be approximated 
to, or separated from, one another. For cover-glasses of all thicknesses 
that position of the correction-screw must be determined at which the 
clearest microscopical image is obtained. The correction is, as a mat- 
ter of course, superfluous with the homogeneous immersion. 



THE MICROSCOPE. 7 

the drop of distilled water upon tlie cover-glass from 
flowing over the edge, and mingling with the fluid in 
which the preparation is mounted. 

This difficulty is absent in the case of cemented 
mounted specimens ; the drop is then very easily re- 
moved from the cover-glass, by means of a fine capil- 
lary glass tube. An oil-drop, on the other hand, can 
only be completely removed by vigorous rubbing. 
We may generally content ourselves with getting rid 
of the superfluous oil by drawing fine blotting-paper 
gently over it, and may leave the very thin layer 
which remains upon the cover-glass. 

3. Eye-pieces. Accessoey Appakatus. Combina- 
tions. — Two eye-pieces are generally used, a weaker 
one for ordinary work, and a stronger for special 
cases ; one of these contains the micrometer. Of the 
accompanying pieces of apparatus, which are furnished 
by the optician, the following may be mentioned : 

(1.) A revolving apparatus for rapidly changing 
the objectives. 

(2.) An arrangement for drawing, preferably Ober- 
hauser's model, which has a knee-shaped bend, and is 
provided with two prisms. 

(3.) A polarizing apparatus. 

(4.) A spectroscope. 

The last two pieces of apparatus have been re- 
cently combined by Messrs. Schmidt and Haensch of 
Berlin. 

(5.) A warm stage — Schultze's or Strieker's.* 

* There follows a list of German opticians whose instruments are 
recommended ; this has "been omitted as being of no practical use to the 
English reader. — Trans. 



II. 

ACCESSORIES. 

1. Illuminating Lamp. Cobbleb's Globe. — Day- 
light always serves best for illumination, especially if 
it can be obtained from a white cloud which is some- 
where in the neighborhood of the sun. Direct sun- 
light can not be used, therefore it is always most ad- 
vantageous to place the working-table at a window 
which faces the south. If the sun is shining, a window 
is darkened by means of a white curtain, under the 
shade of which the work is carried on ; the light is 
derived either from the curtain, or from the sky 
through the uncovered windows. 

However, our clime, which so abounds in cloudy 
and dark days, frequently compels us, even during 
the day, to have recourse to artificial sources of light, 
especially for high powers. We use for this purpose 
merely a gas-flame, with an Argand burner, over which 
is placed an isinglass chimney, and a shade made of 
stiff paper. The direct light of the flame is used, and 
its yellow color is corrected by adjusting over the 
eye-piece a ring containing plane blue glass. 

"When we have at hand several of these rings, with 
varying shades of blue, we can make the correction 
for the different distances at which the lamp is placed. 



ACCESSORIES, 9 

The burner is fastened to a stand by a sliding arm, 
and stands at a height of about twenty or thirty cen- 
timetres above the surface of the table ; if the shade 
is properly attached, by means of a wire frame, the 
heat of the flame need be felt little, if any ; and then 
work by gaslight is not much more trying than by 
daylight. Petroleum-light is certainly useful, but one 
should see that the Argand burner has a diameter of 
at least twenty millimetres; the so-called duplex 
burners of corresponding breadth are also valuable. 

I recommend highly the use of a cobbler's globe 
(Schusterkugel), which is filled with a solution of 
sulphate of copper and ammonia, and interposed 
between the lamp and the mirror. A few drops of 
ammonia are added to a solution of sulphate of cop- 
per, till a beautiful blue color results ; by further 
diluting this solution with water, a cloudiness gener- 
ally appears, which is redissolved by adding more am- 
monia. It is very easy to test the proper intensity of 
the color. A beautiful white light is then obtained, 
which falls upon the mirror in parallel rays. 

The light itself must be placed low, and is only 
to be used for the actual microscopical work; for 
preparing specimens, etc., some other illumination 
must be employed. 

2. Glass Appaeatus. — The slides are to be made 
of white, plane glass, as free from flaws as possible, 
and of a definite, even thickness and size ; the Eng- 
lish are the best. The edges must be ground off. 

The cover-glasses are likewise of a definite, mode- 
rate thickness, preferably about 0'15 millimetres. 
Many strong objectives require cover-glasses of spe- 



10 THE USE OF THE MICROSCOPE. 

cial thinness. Among other glass objects tlie follow- 
ing are used : 

A large number of watch-glasses. 

Glass rods. 

Glass tubes, capillary and otherwise. 

Glass flat-bottomed dishes of different sizes. 

Bell-glasses of different sizes. 

Glass beakers, injecting-bottles, crucibles, reagent- 
glasses, and a stand with funnels. 

Measuring-glass. 

A plate of black glass and one of white porcelain, 
to serve as supports while preparing specimens. The 
black plate is adapted for white or unstained objects, 
while we always carry on our manipulations with 
stained preparations upon the white ground. 

3. Metallic Lststkumejnts. — As regards the me- 
tallic instruments, needles, forceps, scissors, knives, 
spatula, etc., attention can not be directed too strong- 
ly to the fact that these must always be kept in an 
absolutely perfect condition. Even if we do see 
many older microscopists working with blunt, rusty 
needles, with dull scissors, with forceps which do not 
hold, etc., let this only serve as an example to deter 
us from a similar procedure. Our instrumentarium 
must be kept bright and sharp, like an oculist's case. 
The razor has its under surface ground flat ; here, too, 
it is self-evident that the blade must always be sharp 
and clean. In spite of our double knife and micro- 
tome, we still need the razor very often, especially in 
hasty investigations. In cutting, draw rather than 
press, and utilize the whole length of the cut from 
beginning to end. 



ACCESSORIES. 11 

Within a few weeks nearly every one acquires tlie 
requisite experience, so that lie can cut with the razor, 
rapidly and surely, a few even, thin sections, from 
fresh, as well as from hardened, preparations ; in many 
cases, where absolutely exact or very thin sections 
are not required, this method, because of its simplici- 
ty, is always the best. 

The double knife, also, is much used, especially 
for the examination of fresh preparations; with it 
large and regular sections are obtained, even from 
fresh, soft organs. However, the entire gross speci- 
men is usually sacrificed in this way ; the organs are 
frequently hacked to pieces with the double knife 
in a very ugly manner. The instrument is drawn 
quickly through the organ which is to be cut; in 
order that the blade may not strike anything at the 
same time and be injured, it is advisable to use a soft 
support, as fresh liver. 

Most instrument-makers make the springs between 
the two blades of the double-knife too strong ; I have 
often found it advantageous to remove these springs 
altogether. 

The blades are thus adjusted: the upper screw 
is first screwed entirely back, then the two blades 
are closely approximated by the lower screw, and 
finally they are again separated a little, by advanc- 
ing the upper screw. The blades should be nearly 
parallel. 

4. Miceotome. — No microscopist will be willing 
to work, at the present day, without a microtome. 
Although ten years ago microtomes were only in the 
hands of a few, now they are very widely used, and 



12 THE USE OF THE MICROSCOPE. 

it is certain that with, them a most important advance 
has been made in microscopical technology. 

It would be extremely tiresome (and, indeed, it is 
unnecessary) to introduce here a description of the 
various forms of microtomes, since several new mod- 
els or modifications are annually invented. We can 
use almost any system ; however, I w T ould advise 
against those models in which it is necessary to im- 
bed the preparation. Good microtomes are furnished 
by Dr. Long, of Breslau, Katschs, instrument-maker 
in Munich, and by the mechanicians Schanze, in 
Leipzig (at the Pathological Institute), Jung, in 
Heidelberg, and Meier, in Strasburg, and also by 
several others. The author worked for a long time 
with Long's instrument ; but he is so well satisfied 
with Schanze's new model, that he would especially 
recommend this one. The apparatus explains itself : 
the knife is carried on a slide, while the specimen is 
gradually raised by the coarse movement of a toothed 
wheel ; by drawing forward the knife, a section will 
be cut corresponding in thickness to the amount of 
elevation of the specimen. 

In Long's microtomes, the specimen is raised by 
the limited movement of a slide upon a sloping 
plane ; in Jung's instruments it is effected in a very 
accurate manner by means of a fine micrometer-screw. 
In these instruments, also, the sliding movement is 
remarkably smooth. 

The specimen which is to be cut is secured in a 
clamp; clamps of different sizes and shapes, such 
as can be readily changed, may be used with the 
same instrument. In every case, the essential thing 



ACCESSORIES. 13 

is that the specimen should be tightly held in the 
clamp. 

It is customary to secure the preparation be- 
tween two slices of well-hardejied liver ; amyloid liver, 
which has been kept in alcohol, is best fitted for 
this purpose. The thoroughly hardened specimen — 
a firm, even consistence is a necessary prerequisite for 
the making of good microtome sections — is placed in 
the clamp, between the two slices of liver, and is se- 
cured, by means of the screw, in such a way that a 
layer from one to two millimetres in thickness pro- 
jects above the clamp. The slices of liver act like 
fixation-splints, by which the portion of the specimen 
which extends above the clamp is sufficiently secured. 
The fixation must always be perfect ; if the specimen 
can slip away from the cutting-blade only a little, the 
sections are imperfect and unreliable. 

"We may, instead of this, fasten in the clamp a 
cork, upon the upper surface of which a slice of the 
hardened preparation, several millimetres in thick- 
ness, has been glued. As a glue, a thick solution of 
gum or so-called fluid cement is employed. Both sub- 
stances soon become perfectly solid when placed in 
alcohol. In this way only a small piece of the speci- 
men is used, and this is subject to no pressure, since 
it is merely attached to the cork. This procedure, 
which, as far as I know, originated with "Weigert, is 
a very efficient one, and is to be highly commended. 

When the specimen has been properly secured, 
the knife is so adjusted that it commences to cut at 
the point where the edge begins. This direction is, 
indeed, self-evident, but it is very frequently disre- 



14 THE USE OF THE MICROSCOPE. 

garded ; the novice often pushes the blunt part of the 
blade against the specimen, or, on the other hand, lie 
allows the edge to begin cutting at its middle. Both 
of these errors must be avoided. Then care must be 
taken that the knife meets the specimen at the most 
favorable angle. It should be pressed down as little 
as possible, but should have mainly a drawing move- 
ment ; it is, therefore, to be so adjusted, with refer- 
ence to the breadth of the specimen, that the entire 
edge is used up to its very end. The narrower the 
object, the more oblique is the knife, and the more 
economically can the section be made. In cutting, 
the specimen and the knife-blade are always kept 
thoroughly moistened with alcohol ; the slide is to be 
well oiled, preferably with bone-oil. 

Instead of the clamp, which is designed for hard- 
ened specimens, a freezing-plate or freezing-box may 
be attached to the same microtome ; an ether-spray is 
thrown against the under surface of this, by means 
of an atomizer, an intense cold being produced by its 
evaporation. 

A slice of any fresh organ, when placed upon the 
upper surface of the plate, freezes at once; in this 
way very delicate and even sections can be obtained 
directly from fresh organs, "without previous harden- 
ing. 

Thick and Thin Sections. — The thickness of the 
sections can be varied at pleasure, according as the 
specimen is more or less elevated; this thickness can 
generally be estimated directly from the micrometer- 
scale in hundredths of a millimetre. 

The beginner generally thinks that in all cases the 



ACCESSORIES. 15 

thinnest possible sections must be prepared, while a 
practiced investigator often purposely works with 
quite thick sections. Very thin sections have the 
following disadvantages : 

1. They are difficult to manipulate; it often takes 
a good deal of time to spread them out perfectly. 

2. It frequently happens that the elements, in- 
closed within the meshes of extremely thin sections, 
fall out ; this is highly disadvantageous for our pur- 
poses, since the very objects which interest us are 
often lost. Accordingly the process of penciling, or 
shaking, specimens, which is often practiced in normal 
histology, by which thick sections are rendered trans- 
parent, is to be recommended only to a limited extent 
in pathological examinations. 

3. If we are searching for elements which are only 
sparingly distributed through an organ — for example, 
animal or vegetable parasites — there is naturally a bet- 
ter chance of finding the same in thick sections, pro- 
vided that the latter are sufficiently transparent, and 
that the elements sought for stand out well from the 
surrounding tissue. 

4. In thick sections definite stereometric represen- 
tations of the structure of the object in question are 
frequently obtained, since a number of sections placed 
one above the other are examined directly in situ and 
in continuo, while in extremely thin sections plane 
images only are formed. On the contrary, it is evi- 
dent that some particularly delicate structures gener- 
ally become visible only in thin sections, while in 
thicker ones, in consequence of the many superim- 
posed outlines, they completely disappear. 



16 THE USE OF THE MICROSCOPE. 

For most purposes sections of fresh organs hav- 
ing a thickness of 0'05 mm., or even 0*1 mm., are 
very useful; in the case of hardened preparations 
thinner ones are generally used, especially if they are 
stained, about # 01 to 0*0 3 mm. being the average. 

It is a very important advantage of microtomes 
that with them sections of any desired thickness can 
be produced easily and in any number, as if from a 
manufactory. More sections are usually made than 
are needed at the time ; the rest are preserved for 
future use, in a small bottle which is filled with al- 
cohol. 

Further Treatment of Sections. — The sections 
are transferred from the microtome-blade (or from 
the razor or double knife) to a watch-glass filled with 
fluid, and this is best done by means of a soft, moist 
brush. The fluid used for fresh (frozen) sections is 
salt-solution, and in this they are examined directly ; 
for sections made from alcoholic specimens, first alco- 
hol, and then, as a rule, distilled water are used. 
The sections may easily be ruined during the further 
manipulations to which they are subjected ; to allow 
of the action of reagents and staining-materials they 
must frequently be transferred from one watch-glass 
to another, and finally be placed unharmed upon the 
slide. Slightly bent, thin spatulas are best used for 
this purpose — for example, strips of copper or pew- 
ter, or even of nickel-plated steel, which are brought 
carefully under the object as it floats in the fluid ; 
in this way alone can a thin section be lifted out 
without wrinkling and be placed in a new fluid. 

The slide also must first be covered with a thick 



ACCESSORIES. 17 

layer of fluid, before the section is transferred to it ; 
the section should slip easily from the spatula, sim- 
ply through the action of a gentle current of fluid. 
The cover-glass is then placed upon it, and the excess 
of liquid is absorbed by means of a capillary tube, or 
by blotting-paper. The slide rests meanwhile upon 
a black glass plate, in the case of unstained objects, 
or upon a white plate, when the objects are stained. 
The microscopist should also keep both hands free ; it 
is a very bad habit with many beginners to hold the 
slide in the hand while placing the specimen upon it. 

In order to render possible the delicate move- 
ments of the fingers, which are necessary for these 
and other manipulations, I advise that the fore-arm, 
and also the ulnar edge of the metacarpus, should be 
firmly supported upon the table ; it is veiy difficult 
to mount thin sections properly when the arm is un- 
supported. 

The sections prepared from alcoholic specimens 
are transferred from the watch-glass containing alco- 
hol to distilled water ; by reason of the active move- 
ments which are caused by the currents of diffusion, 
they spread out very nicely here, and become changed 
from their shrunken, wrinkled condition into transpar- 
ent laminae. Then for the first time do they become 
fit for examination ; they are arranged upon the slide 
in distilled water, which is then in most cases dis- 
placed by glycerin, that has been placed at the edge 
of the cover-glass. Large, delicate sections do not 
unroll so well in the viscid glycerin as in water ; 
smaller sections may also be placed at once in a drop 
of glycerin. 



III. 

REAGENTS. MICRO-CHEMISTRY. 

Reagents are to be kept in small glass bottles 
with ground-glass stoppers. While this principle is 
carried out in every chemical laboratory, and even in 
every pharmacy, we constantly see many microsco- 
pists working with bottles which have dirty corks- — 
a most reprehensible custom. Even a double stop- 
per is proper for those reagents which are constantly 
used ; we always keep our glycerin, acetic acid, dis- 
tilled water, oil of cloves, Canada balsam, etc., in so- 
called cobalt-flasks, the ground-glass stoppers of which 
are drawn out below into a glass tube, while a hat- 
shaped glass cover is placed over the whole. The be- 
ginner should, by preserving his reagents in an abso- 
lutely clean and transparent condition, become accus- 
tomed, from the very first, to the most painstaking 
care in his work. 

We, as a matter of course, alway employ prepa- 
rations which are chemically pure ; as yet we have 
never used any substances, except certain dyes, which 
can not be purified chemically. In order to insure 
cleanliness, besides the method of preserving reagents 
just mentioned, it is furthermore desirable that we 
should not use them in an uncleanly manner ; he who 



REAGENTS. MICRO-CHEMISTRY. 19 

dips a teasing-needle, brush, or even his finger, into his 
reagents, in order to remove a drop, will never be fit 
to undertake the more delicate investigations, espe- 
cially the examination of schizomycetes. 

Only a carefully-cleansed glass rod, or a glass tube 
which has been freshly heated, should be brought in- 
to contact with the reagent. 

" Artificial Products." — The use of reagents is 
of the highest importance in histological examina- 
tions ; many structural elements can only be studied 
by the aid of chemical influences. We, of course, 
endeavor to examine objects when they are as little 
changed as possible, in their normal — if possible in 
their living — condition ; however, it would be a great 
mistake to reject as " artificial products " all the struct- 
ures which can only be rendered visible by the use of 
certain reagents. No signs of a nucleus can be seen, 
as a rule, in the living white blood-corpuscle, and no 
cells in the living cornea, because the differences in 
the optical behavior of the nucleus and the proto- 
plasm, and of the cell and the basement-substance, 
are too small to become visible, or rather, because 
the surrounding substance is too opaque to allow 
the delicate outlines of the inclosed body to appear 
through them. When death ensues, on account of 
various chemical changes, as coagulation, etc., these 
differences become more marked — that is, the envel- 
oping substance becomes more transparent, and we 
are, at all events, justified in assuming that the nu- 
cleus in the living white blood-corpuscle, and the 
cells in the living cornea, are already present, although 
we are not able to demonstrate them until after post- 



20 THE USE OF TEE MICROSCOPE. 

mortem change has begun ; this is precisely the case 
with the borders of many epithelial cells, with the axis- 
cylinders of nerve-fibres, etc., which are also invisible 
in the living state, but are none the less present. 
Besides this, in examinations in pathological anatomy, 
we almost never observe the tissue in its living, nor- 
mal condition, but always as the seat of more or less 
advanced cadaveric change. We must, accordingly, 
always bear in mind that a structure which is found 
regularly was not necessarily present as such intra 
vitam ; however, it does point in every case to a dif- 
ferentiation which existed during life, and which is 
brought out by the cadaveric change, or by the 
reagent used. 

Furthermore, if among a certain number of ele- 
ments, which originally appeared to he identical, some 
conduct themselves in a peculiar way toward a certain 
reagent, while others do not; if, for example, some are 
stained by a certain dye, while others remain colorless, 
we must necessarily conclude that there was a primi- 
tive difference between the elements. 

Upon this principle are founded all of the meth- 
ods of preparation, some of them very complicated, 
which are employed for the exhibition of the different 
histological elements. From this simple observation 
it is evident what attitude we have to assume toward 
the microscopical images that are produced by our 
reagents. 

We must not be confused by the expression 
" artificial product " ; in former times many impor- 
tant histological discoveries were at first discredited 
in this way. 



BE A GENTS. MICRO- CHEMISTS Y. 21 

In our microscopical investigations, we do not, as 
a rule, act simply as observers, but we experiment, 
and our results are consequently made up of pre- 
formed objects on the one hand, and of factors intro- 
duced by ourselves on the other. 

He would certainly be ever liable to tlie gross- 
est errors, who should decide upon the question of 
original structure simply from his own results, with- 
out regard to these relations, which are generally 
very simple; an apparent liber, for example, may 
either correspond to a genuine pre-existent fiber, or 
may represent a fold, or a production of coagulation, 
etc. 

In our pathological examinations especially, we 
employ reagents for still another purpose. We often 
have the task of searching for certain elements, for- 
eign bodies, parasites, etc. ; if, now, we know that 
these elements which are sought for resist certain 
reagents and methods of treatment, while other sub- 
stances are destroyed by this same treatment, then 
we have a very useful method of examination for our 
definite purpose, although the structural relations of 
the organ are entirely lost. 

It follows from all this that microscopy, especially 
if it is concerned with pathological objects, should not 
be regarded as a purely mechanical process, for it fre- 
quently requires a certain amount of care and circum- 
spection, even in the choice of the modus procedendo 

Micro-chemical Examinations. — Micro- chemical 
examinations are so conducted that either the speci- 
men remains for some time in contact with the re- 
agent (for example, it is placed in a watch-glass filled 



22 THE USE OF THE MICROSCOPE. 

with the reagent, and is then examined under the 
microscope), or in such a way that the reagent acts 
upon the specimen while the latter is actually under 
the microscope. With this object, the reagent is 
placed at the edge of the cover-glass, and gradually 
makes its way toward the specimen ; we can hasten 
this process by absorbing the fluid with blotting- 
paper at the opposite edge of the cover-glass. In 
this way the influence of the reagent may be directly 
observed under the microscope — for instance, the so- 
lution of granular protoplasm, of the red blood-cor- 
puscles, and of lime, under the action of acids, etc. 
The beginner must naturally be extremely careful 
that the reagent does not get upon the upper surface 
of the cover-glass ; in that case the object-lens of the 
microscope might very easily be injured. Other com- 
plicated reactions, especially most of the staining 
processes, are usually undertaken in watch-glasses; 
after having been acted upon by the reagent the 
specimen is compared with a model, or the same ob- 
ject is sketched first before, and then after, it has 
been treated. 

The reagents principally employed are the follow- 
ing: 

1. Distilled Watek. — Distilled water, as a rule, 
still contains small quantities of dissolved substances, 
and it furnishes (especially in summer) a favorable 
nidus for various minute organisms. If, therefore, 
micro-organisms are found in a specimen which has 
been treated with distilled water, this error must be 
guarded against ; they can easily be removed by re- 
peated boiling. The water diffuses at once, and very 



REAGENTS. MICRO-CHEMISTRY. 23 

actively, into most portions of the fresh tissues ; in 
every case the vital properties of those elements of 
the human body which have been isolated in dis- 
tilled water very soon vanish. The dead cellular 
elements, removed from the cadaver, are likewise es- 
sentially altered ; the most rapid change takes place 
in the red blood-corpuscles; they swell, discharge 
their coloring-matter, and soon become perfectly in- 
visible. 

The method of employing distilled water in the 
examination of fresh tissues, and its limits are briefly 
as follows : We use it by preference in those cases in 
which we desire for our purpose to remove quickly 
from substances very rich in blood the blood-corpus- 
cles, which, on account of their large number, fre- 
quently trouble us by concealing the other ele- 
ments ; however, we must never forget that the tis- 
sue itself may at length be essentially changed by the 
water. 

If we are working with alcoholic specimens, dis- 
tilled water simply causes them to swell, and gener- 
ally in a symmetrical manner, so that nearly the orig- 
inal gross relations are restored. It is only seldom 
that further changes are induced in this instance, 
since the essential parts of the tissues, the albumin- 
ous bodies, are coagulated — that is, they are trans- 
formed into a variety that is insoluble in distilled 
water. However, we must always keep in view the 
fact that the diffusible substances that are soluble 
in water, as glycogen and sugar, are quickly removed 
from the sections. 

2. Salt-solution of 0*8 per Cent. Ikdiffee- 



24 THE USE OF TEE MICROSCOPE. 

eotd Fluid. — The most varied micro-organisms very 
soon develop in this in great numbers ; the fluids 
must therefore be very often renewed. I strongly 
advise against adding antimycotic substances, since 
we then no longer have a pure salt-solution ; on the 
other hand, the solution may easily be sterilized by 
boiling. 

In order to preserve the protoplasm and the red 
blood-corpuscles as nearly intact as possible, a salt- 
solution of 0'8 per cent is employed, the usual fluid 
in pathological examinations, which is suitable for 
sections of fresh tissues, as well as for the dilution of 
liquids. If we are particularly interested in preserv- 
ing the vital properties of the cells for a longer pe- 
riod, we add to nine parts of the salt-solution one 
part of egg-albumen — the so-called artificial serum — 
or we use aqueous humor, hydrocele-fluid, transuda- 
tions, blood-serum, etc. 

3. Absolute Alcohol. Hakdenlkg. — Always 
use the purest alcohol possible, and never the ordi- 
nary spirit, which, in addition to the water that 
it contains, is always contaminated with other sub- 
stances, and frequently even has an acid reaction. 
In case we desire to use dilute alcohol, the absolute 
alcohol is mixed with the necessary amount of dis- 
tilled water. 

Alcohol diluted with two parts of water is some- 
times used as an agent for the isolation of tissue- 
elements (Eanvier) ; while the cells become capable 
of resistance when placed in it, the cementing sub- 
stances remain soft, so that the isolation of cells, 
which seemed previously to be fused together and 



REAGENTS. MICRO-CHEMISTRY. 25 

to be attached to the intercellular substance, is an 
easy matter. To this end the bits of fresh tissue are 
immersed for about twenty -four hours in thirty-three 
per cent, alcohol. 

The principal use of alcohol, however, is in the 
hardening of tissues, so as to give them a proper con- 
sistence for cutting. The process of hardening de- 
pends essentially upon two factors — the abstraction 
of water, and the coagulation of albuminates ; beside 
this, alcohol removes from the pieces of the organ 
certain extractives, which are of no morphological 
importance, and a small amount of fat. 

The immediate inference from this is that in all 
cases we can form a correct conclusion regarding the 
amount of fat in tissues (in pathological fatty degen- 
erations, for instance) only by the examination of 
fresh organs, and never in alcoholic preparations. 

Through the abstraction of water there naturally 
occurs a diminution, or shrinking of the specimens ; 
if the different portions of the same object con- 
tain unequal amounts of water, the shrinking will 
take place in an uneven manner, and the specimen 
will be deformed in a very undesirable way. How- 
ever, the form is generally quite well preserved, and 
corresponds to the diminution of the preparation due 
to the action of the alcohol ; the sections swell up 
again if they are immersed in distilled water (through 
the absorption of water), so that they then resemble 
very closely their original condition. The main dif- 
ference consists in the opacity of the sections, occa- 
sioned by the granular, coagulated albuminates, and 
this remains even after they have become swollen in 



26 THE USE OF THE MICROSCOPE. 

water ; as a remedy for this evil we generally use 
glycerin (q. v.) as an optical clearing-agent, or even 
acids and alkalies, by which the precipitated albu- 
minates are redissolved, although many structures 
are destroyed at the same time. Hardening in alco- 
hol is best effected by placing small fragments of 
organs in large quantities of absolute alcohol ; in this 
way it is evident that the piece is entirely surround- 
ed by the fluid. 

A piece two or three cubic centimetres in size 
can thus be thoroughly hardened within twenty-four 
hours, and smaller bits still more rapidly. 

The method, formerly in frequent use, of im- 
mersing the organs first in weak alcohol, and then in 
that of gradually increasing strength, has properly 
been altogether abandoned. 

Alcohol is the best hardening-fluid for most tis- 
sues ; * we employ it almost exclusively for this pur- 
pose. In the case of morbid specimens, it is of es- 
pecial importance to us that the changes in their 
substance, occasioned by the method of preparation, 
should be simple and such as can be readily con- 
trolled ; this is the action of alcohol, while the pro- 
cess of hardening in chromates, formerly so popular, 
which varies according to the differences in time, 
temperature, etc., causes changes, as clouding and stain- 
ing, which it is very difficult to obviate. 

A few little devices, which sometimes become 
necessary, are almost self-evident; for example, the 
eye is apt to shrivel up very quickly when placed in 

* As has already been mentioned, hardening in alcohol is not adapt- 
ed for examinations of fatty tissues. 



REAGENTS. MICRO-CHEMISTRY. 27 

alcohol. This condition can easily be remedied by 
injecting the fluid into the vitreous humor with a hy- 
podermic syringe, during the early part of the hard- 
ening process, until the firmness and roundness of the 
globe are restored. This process must subsequently 
be repeated. 

Many tissues, such as lung, muscle, etc., do not 
acquire a proper consistence for cutting, even after a 
long stay in alcohol ; * it is advisable in these cases 
to immerse the imperfectly hardened specimen for 
twenty-four hours in thin mucilage — equal parts of 
mucilage and glycerin. If the specimen saturated 
with solution of gum is again placed in alcohol, it 
hardens very evenly and firmly, since the gum is pre- 
cipitated by the agent. The gum is very soon dis- 
solved out of the sections when they are immersed in 
water. For the central nervous system alone, at least 
for the white substance, alcohol is not well adapted. 
In this instance its hardening effect is imperfect, cor- 
responding to the smaller amount of water ; besides 
this, alcohol abstracts a large part of the fatty mat- 
ters of the nerve-medulla, which are then precipi- 
tated in a crystalline form, so that the tissue is great- 
ly injured. 

I have never been able to dispense with the 
chromates in the treatment of these important or- 
gans. 

4. Ether. Chloroform. The Removal of Fat. — 
Both substances are frequently employed in order to 
remove fats. As a matter of course they do not act 

* This is sometimes the case with rather old alcoholic preparations 
which are intended to be further hardened. 



28 THE USE OF THE MICROSCOPE. 

upon fresh tissues, since these are saturated with wa- 
ter, and chloroform and ether do not mix with water. 
The pieces (or sections) of an organ must first be 
dehydrated by treating them for some time with 
alcohol. The section from which fat is to be re- 
moved is then placed for about five minutes in a 
watch-glass filled with absolute alcohol, and then in 
a watch-glass containing ether or chloroform ; if the 
fluid becomes clouded, it is a sign that the section is 
not yet sufficiently dehydrated, and it must be again 
placed in absolute alcohol. When it has remained 
for a few minutes in ether or chloroform,* so that 
the substances soluble in it are entirely removed, the 
section is transferred to alcohol for some time, and 
then to a watch-glass filled with w r ater. It is exam- 
ined either in water, or, as a rule, on account of the 
extreme clouding, due to the coagulation of the albu- 
minates, acetic acid must be added in order to redis- 
solve the albuminates. The order which is followed 
in the process of removing fat from fresh tissues is 
this : 

Salt-solution or water. 

Alcohol. 

Chloroform or ether. 

Alcohol. 

Water to which acetic acid is added. 

5. Acids, (a) Sulphuric, Hydrochloric, and Ni- 
tric Acids; Decalcification. — The strong mineral 
acids, when highly concentrated, possess the prop- 

* In chloroform the sections become at once quite transparent ; this 
does not depend upon the solution of the fats, but is a simple physical 
phenomenon, due to the high index of refraction of chloroform. If 
returned to alcohol the section immediately shows its former opacity. 



REAGENTS. MICBO-CHEMISTRY. 29 

erty of rapidly coagulating albuminous bodies ; they 
can, in consequence, be used with advantage for fixing 
certain very delicate structures, as the so-called nu- 
clear figures. According to Altniann,* it is best to 
immerse the pieces of organ for a short time (about 
one second) in a three-per-cent. solution of nitric acid, 
having a specific gravity of 1020, then to wash them 
in distilled water, and to harden in absolute alcohol. 
Flemming and other authors use a still stronger solu- 
tion of nitric acid for the same purpose. When con- 
siderably diluted (about 1 to 1,000), the mineral acids 
cause essentially swelling of those substances which 
contain the most protoplasm (such as contractile or 
gelatinous tissue), the same as acetic acid. 

The same reagents are used also to remove lime- 
salts. In order to make good sections through cal- 
careous parts, such as bones, teeth, calcified tumors, 
etc., the lime must be removed ; we seldom employ 
the earlier method of examining thinly-ground sec- 
tions. This removal is accomplished most quickly 
by means of dilute hydrochloric or nitric acid. A 
one-half per cent, solution of the acid is used, and this 
is mixed with alcohol containing sodium chloride, in 
order to avoid the swelling of the basement-substance. 
The formula is as follows : 

Hydrochloric acid . . 5 parts. 

Sodium chloride ... 5 " 

Distilled water . . . 200 " 
Alcohol 1,000 u 

The decalcifying fluid must be frequently changed, 
as it then gives quite brilliant results. 

* Altmann, " Arch, fur Anat. und Physiol.," 1881. S. 219. 



30 THE USE OF TEE MICROSCOPE. 

A solution of chromic acid (about one per cent), 
or a saturated solution of picric acid, acts rather more 
slowly (Ranvier). 

Moreover, we often find in a microscopical prep- 
aration deposits darker than the surrounding tissues, 
which lead us to suspect that we have to do with 
lime. "We are convinced that they are lime if we 
observe that their dark outline disappears on adding 
an acid ; in most cases the lime is combined with car- 
bonic acid, so that on the addition of acid there occurs 
an active development of gas, presenting under the 
microscope a very elegant and striking picture. 

If sulphuric acid is used, sulphate of lime, or gyp- 
sum, is formed, which is soluble with difficulty and 
crystallizes rapidly, in beautiful prisms, often uniting 
to form tufts. Gypsum-crystals are exquisitely doub- 
le-refracting, as we can prove at once by means of a 
Nicol's prism attached in front of the eye-piece. 

Many cementing-substances are soluble in strong 
acids; for example, a twenty-per-cent. solution of 
hydrochloric acid, or a thirty -three-per-cent. solution 
of liquor potassse, is used to isolate smooth muscle- 
fibres. In order to isolate the urinary tubules over 
a wide area, so as to demonstrate their complicated 
course, strong hydrochloric acid is employed, with the 
application of gentle heat ; however, this method has 
not yet come into use in the study of nephritis. 

(b) Acetic Acid. — Organic acids, especially acet- 
ic, are very often used in our work ; they are prin- 
cipally employed to dissolve, or to cause to swell, the 
albuminates and the gelatinous substance of which 
the connective-tissue fibrils are known to consist. 



REAGENTS. MICRO-CHEMISTRY. 31 

Since the substance of nuclei and elastic tissue, fats, 
the medulla of nerves, etc., resist acetic acid, this is a 
very convenient agent with which to expose the nu- 
cleus that is concealed within a darkly-granular cell, 
and the elastic tissue that is distributed throughout 
the connective tissue, in the substance of muscles. 
By the action of acetic acid also the fat-granules, 
deposited in protoplasm, in the contractile substance 
of muscles, etc., appear much more clearly. The 
micro-organisms which are present in the tissues be- 
have in the same way. 

Acetic acid acts in the manner described, even in 
a dilute solution of 1 to 100 ; in a solution of 1 to 
1,000, also, its clearing action is still apparent, only it 
is somewhat slower. 

If a section from a fresh organ, or from an alco- 
holic specimen, be placed in acetic acid, in a watch- 
glass, it generally becomes quite transparent, and at 
the same time it swells considerably ; this swelling 
usually takes place in an uneven manner, so that the 
section assumes a coarse, wavy character. It thus 
becomes nearly useless for examination, It is better 
to allow the acetic acid to act upon the sections be- 
neath the cover-glass ; a momentary raising of the glass, 
so as to allow the air to get under it, is sufficient to 
permit the drop of acetic acid, which has been placed 
at its edge, to make its way beneath, while the slight 
pressure of the cover is enough to preserve the even 
shape of the section. If the action of the acetic acid 
is to be rapid and energetic, the undiluted acid (glacial 
acetic) is used ; for most purposes, however, it is ad- 
visable to dilute it with a little distilled water. 



32 THE USE OF THE MICROSCOPE. 

In many substances, which are saturated with al- 
kaline albuminous fluids, acetic acid at first causes a 
clouding, due to the neutralization of the alkali ; if 
more acid be added, this cloud clears up. How- 
ever, a permanent opacity may be occasioned by 
acetic acid, which is not cleared up by excess of the 
acid ; that is, mucin is precipitated by it. Fibrin, 
serum-albumin, and mucin are often found together 
in exudations, in the contents of cysts, etc. The ac- 
tion of acetic acid will vary according to the quan- 
titative relations of the mixture ; in most cases sub- 
stances are rendered very clear and transparent by it. 

With the marked swelling that acetic acid occa- 
sions in albuminous and gelatinous bodies, it is not 
surprising that the outlines which appear after the 
action of the acid, as well as the borders of the 
structures that resist it, do not always remain 
entirely unchanged. As far back as 1840 Henle 
called attention to the fact that the several nuclei 
which appear in white blood-corpuscles, pus-corpus- 
cles, etc., after being treated with acetic acid, were 
not preformed, but that these contained only a single 
nucleus, which through the action of the acid was 
broken into several pieces. This actually occurs in 
many cases. Although we now know that some of 
the lymphoid cells, even in the living state, possess 
several nuclei, it is, nevertheless, highly important to 
remember that the nuclei that appear in a cell after 
it has been exposed to the influence of acetic acid 
may possibly represent artificial products, or frag- 
ments of a nucleus originally single. 

In connective tissue acetic acid often causes an 



REAGENTS. MICRO-CHEMISTRY. 33 

essential change of structure ; for instance, the reg- 
ular rows in which the cells of tendons and fascia 
are arranged, are not usually to be distinguished 
after the swelling due to acids, while the nuclei are 
apparently strewn about in disorder. Ranvier took 
the precaution to fix upon a slide a small tendon, 
which was kept in a state of tension by means of 
little balls of wax applied to its ends, to cover it, 
and then to allow acetic acid to act upon it slowly ; 
the swelling then occurs less irregularly, and the ar- 
rangement of the nuclei in rows becomes clear. 

The beginner is strongly advised to convince him- 
self of these and similar facts by personal experiment, 
in order that he may be in a position to decide what 
change he is inducing in structures by the addition of 
different fluids. 

Formic and tartaric acids are less used ; their ac- 
tion is similar to that of acetic acid. 

(c) Picric Acid. — Picric acid, on the contrary, has 
a special use — namely, as a hardening and a staining 
material ; the albuminates are gradually transformed 
in a saturated solution of picric acid into the insolu- 
ble variety, so that the tissues, with hardly any 
shrinking, assume a consistence proper for cutting. 

Most substances are stained yellow at the same 
time, some very intensely, as smooth muscles, the 
horny cells of pavement epithelium, and of the epi- 
dermis, etc. 

This characteristic staining also occurs very beau- 
tifully in sections which have been made from alco- 
holic specimens, and, indeed, in a very short time, 
within a few minutes; however, it is soon soaked 



34 THE USE OF THE MICROSCOPE. 

out again by the action of water and alcohol. If we 
desire to preserve the staining, a small quantity of 
picric acid must be added to the water, alcohol, or 
glycerin. 

(d) Chromic Acid ; Chromates / Mullens Fluid. 
— Chromic acid, when very dilute (about 1 to 10,000 
or 1 to 20,000), is used as a macerating-fluid ; if a 
small piece of spinal cord, for example, be placed 
in such a solution for twenty-four hours, it is then 
very easy to isolate the ganglion-cells with their 
numerous branching processes, while the cementing- 
substance is softened or dissolved. The action of 
chromic acid as a hardening agent is more important 
for our purposes; we use either a solution of the 
acid (having a strength of from one fifth to one per 
cent.), or else its salts. The principal ones in use are 
the bichromates of potassium or ammonium in a solu- 
tion of about two per cent. ; Muller also added sul- 
phate of sodium to a solution of the bichromate of 
potassium. The following is the formula for Muller's 
fluid : 

Bichromate of potassium . 2 parts. 
Sulphate of sodium . . 1 part. 
Distilled water . . . 100 parts. 

It is much used as a hardening-fluid for the nerv- 
ous system and the eye. 

Perfect hardening occurs very slowly, only in the 
course of weeks and months, and the more slowly 
the larger the immersed pieces are, since the chro- 
mate penetrates gradually into the interior of speci- 
mens ; six months or a year may be allowed for a 
cerebral hemisphere, but it has then a firm consist- 



REAGENTS. MICRO-CHEMISTRY. 35 

ence. The hardening-fluid must be frequently changed, 
and in order to prevent the formation of mold in 
the solution a bit of camphor is added (Klebs). 
According to Weigert the hardening takes place 
much more rapidly if it is carried on in an incubator 
at a temperature of 30° to 40° C. Erlitzki suggests 
a fluid consisting of two and one half per cent, of 
bichromate of potassium and one half per cent, of 
sulphate of copper ; in this preparation hardening 
occurs very quickly (in from eight to ten days) even 
at the temperature of the room. 

The specimens are then placed in alcohol, which 
can be afterward slightly diluted. 

The central nervous system after this treatment 
assumes a very even, firm consistence ; at the same 
time it shows, even to the naked eye, certain charac- 
teristic differences in color. The gray substance is 
distinguished from the white by a brighter staining, 
while the latter becomes dark green ; the usual form 
of gray degeneration, or sclerosis, in the white col- 
umns shows a dark brown shade, while most of the 
secondary degenerations take a brighter staining, 
even in those cases which did not display in the 
fresh state any difference in color between normal 
and degenerated portions. As regards other organs, 
hardening in solutions of chromic acid, or the chro- 
mates, formerly very popular, is to be recommended 
only in rare cases ; except in the case of the nervous 
system and the eye, we much prefer hardening in 
alcohol, supplemented by the process of soaking in 
mucilage. Fibrous, or net-like, coagula are often pro- 
duced by the chromates, and these may be erroneous- 



36 THE USE OF THE MICROSCOPE. 

ly regarded as preformed bodies; furthermore, the 
dark, granular precipitates within cells and interstitial 
tissues, due to their influence, are often quite trouble- 
some, and are very difficult to clear up by means of 
chemical reagents. Deposits of lime are gradually 
dissolved by chromic acid and the bichromates, and 
may thus escape observation. Micro-chemical reac- 
tion can, as a rule, no longer be employed with speci- 
mens which have been treated with chromates ; for 
these and other reasons, the latter are recommended 
as hardening agents only in those cases in which 
alcohol does not act favorably, on account of the 
peculiar chemical composition of the tissue — in fact, 
only in the nervous system, or in organs that are 
very fatty. It is always desirable to watch the re- 
sults in freshly-examined or alcoholic specimens, on 
account of certain lime deposits which, through the 
action of chromic acid and its salts, may readily be 
completely dissolved and escape observation. 

The simple chromate of ammonium, in a five-per- 
cent, solution, was used with great advantage by 
Heidenhain, especially in demonstrating the rod-like 
structure of the epithelium of the renal tubules. 
The agent is also to be recommended in pathological 
examinations. 

6. Alkalies. Liquor Potassjs aint> Liquor 
Soile. Ammonia. — Alkalies cause a breaking up, or 
swelling, of albuminates, of gelatinous material, of 
the contractile substance of smooth and striated mus- 
cles, and of nuclei; even horny structures are ren- 
dered perfectly transparent by these. About the 
only tissue-elements that resist are elastic tissue, fats 



REAGENTS. MICRO-CHEMISTRY. 37 

(including nerve-medulla), lime, pigment, etc., and 
amyloid material, beside cliitin (the hooks of teniae, 
and echinococci), cellulose, threads of fungi, spores, 
and schizomycetes. From this it is at once evident 
how frequently we make use of alkalies ; they come 
into play whenever we are looking for the bodies 
last mentioned. The^ structure of the tissue is, of 
course, almost entirely destroyed. While we can 
always gain a pretty good idea of a section that has 
been perfectly cleared by acetic acid, when alkalies 
are used everything disappears and we have no 
guides except the elastic tissue and the homogeneous 
membranes. Liquor potassse or liquor sodae (in a so- 
lution of from one to three per cent.) is best used for 
most purposes ; the clearing action begins at once, 
even with this dilution. The concentrated lye (thirty- 
three per cent.) has a special action ; in this solution 
most of the elements are preserved, while the cement- 
substance is dissolved. This applies particularly to 
smooth and striated muscular fibers. If, for exam- 
ple, a bit of a uterine leio-myoma is placed for a 
few minutes in a watch-glass filled with a thirty-three- 
per-cent. solution of liquor potassse, it separates into 
its individual fiber-cells under the needle, almost of 
itself ; only care must be taken that the lye does not 
become diluted, for in that case the fibers themselves 
at once dissolve. The preparation must be examined 
directly in the lye. 

Even red blood-corpuscles preserve their shape in 
the thirty-three-per-cent. solution, while they at once 
disappear in dilute solutions. 

According to an observation of Virchow, weak 



38 THE USE OF THE MICROSCOPE. 

alkaline solutions are able to excite the movements of 
ciliated epitlielia after they have become motionless 
and apparently dead. 

7. Glyceric. — Glycerin must, above all, be with- 
out a trace of acid ; a small quantity of water does 
less harm. It is generally used in a pure form, since 
when diluted with water it is very apt to become 
moldy. Glycerin is of great value in the histologi- 
cal examination of organs which have been hard- 
ened in alcohol, and in other fluids that coagulate al- 
bumen, such as picric and chromic acids and their 
salts. During this process the tissues have necessa- 
rily become much clouded ; if acids or alkalies are 
used, in order to dissolve the albuminous granules 
that have been precipitated by the hardening agent, 
many other structures are destroyed at the same 
time, such as connective-tissue fibers, fibrin, and 
blood-corpuscles. 

For these cases, therefore, glycerin is used as a 
clearing agent. The clearing action of this fluid is 
due, not to the chemical solution or swelling of the 
albuminous granules (fat alone is gradually dissolved 
in glycerin), but rather to a physical force, to its 
high refracting power. We can at once demonstrate 
this action to ourselves by comparing the outline of 
a glass rod which is dipped in water with that of a 
similar rod dipped in glycerin; the latter is much 
more delicate. Or, if one piece of filter-paper is satu- 
rated with water, and another with glycerin, the lat- 
ter becomes much more transparent. The outlines 
of a fragment of tissue which has been moistened 
with glycerin are altogether more delicate; glycer- 



REAGENTS. MICRO-CHEMISTRY. 39 

in is therefore useless for the examination of most 
fresh tissues, the elements of which already possess 
delicate outlines, since the latter then become almost 
invisible. On the contrary, the degree of transpar- 
ency effected by glycerin is peculiarly adapted for 
alcoholic specimens. It may be said that since its 
introduction into microscopical technology the ex- 
amination of organs hardened in alcohol has reached 
its full development. Glycerin mixes with water, 
also with alcohol, acetic acid, etc., in all proportions, 
but rather slowly because of its sirupy consistence, 
so that a glycerin-preparation is a suitable one for 
the induction of a rapid chemical reaction, for ex- 
ample, the action of iodine or an acid. However, 
it is very easy to remove glycerin from the prep- 
aration, by simply placing it in a watch-glass filled 
with water. 

Glycerin has also the well-known property of 
neither evaporating when exposed to the air, nor 
undergoing any other chemical changes ; at the most 
it only absorbs a little water, under some circum- 
stances. This property renders it an excellent ma- 
terial for the preservation of microscopical speci- 
mens. If we desire to preserve a preparation which 
is in water, or a watery solution, it is only necessary 
to place a drop of glycerin upon the edge of the 
cover-glass; the glycerin flows under the cover as 
the water evaporates. Fresh preparations may also 
be kept in this way ; if the glycerin is subsequently 
displaced by water or salt-solution, the original con- 
dition is restored. 

The dark shading, and the shining appearance of 

3 



40 THE USE OF THE MICROSCOPE. 

the elastic fibers and laminae, are only slightly dimin- 
ished in glycerin, since their refractive power is con- 
siderably higher than that of the latter ; on the con- 
trary, the characteristic luster presented by amyloid 
material, and by Recklinghausen's so-called hyalin, 
and other colloid bodies, when examined in watery 
fluids, disappears almost entirely in glycerin, since 
their refractive power differs only very slightly 
from that of the latter. On careful examination 
the difference is still somewhat apparent in most 
cases; it is always well to examine the specimens 
first in water, if you are looking for these objects. 
It has already been stated that small fat-drops dis- 
appear entirely in glycerin, hence glycerin-prepara- 
tions should never be used in looking for fatty 
degeneration. 

8. Acetate of Potassium. — A saturated solution 
of acetate of potassium (as recommended by Max 
Schultze) may also be used as a preserving-fluid; 
it does not evaporate, and is stable in the air. It 
has only a feeble clearing power, hence it is to be 
employed especially for keeping fresh objects that 
have not been hardened. This method is quite use- 
ful for preserving fatty degenerated tissues ; how- 
ever, the outlines of the fat-drops and, in time, their 
original distinctness, is lost. 

9. Oil of Cloves. Canada Balsam. — If we 
wish to clear preparations more thoroughly (espe- 
cially after they have previously been stained in- 
tensely), we use oil of turpentine, or, what is to be 
recommended more highly, oil of cloves ; other ethe- 
real oils, such as oil of cedar, origanum, cinnamon, 



REAGENTS. MICRO- CHEMISTRY. 41 

bergamot, anise, etc., also xylol and phenol, act in a 
similar way. 

Every one can select that substance which is least 
disagreeable to his olfactory organs. These fluids 
are either not at all, or only slightly, miscible with 
water, so that the sections that are to be cleared 
are first dehydrated with alcohol ; it is enough to 
place them for a few minutes in a watch-glass con- 
taining alcohol, after which they at once become 
saturated with oil of cloves. The sections thus at- 
tain the highest degree of transparency ; the refract- 
ive power of the fluids mentioned is very great, much 
greater than that of glycerin, and nearly the same as 
that of glass. The most resistant outlines of the ele- 
ments of animal tissues disappear almost completely 
under this treatment ; in unstained preparations near- 
ly everything vanishes, as a rule, and even elastic 
fibers are difficult to recognize, especially if open 
illumination by means of the Abbe-Koch condenser 
is used ; but the stained portions appear so much the 
more distinctly. With this method of examination, 
therefore, we must always keep in view the fact that 
most of the structures have been purposely excluded 
from our view. Specimens thus prepared can at once 
be permanently preserved in resinous mounting-mate- 
rials ; we generally use Canada balsam, or xylol, dis- 
solved in equal parts of chloroform. A solution of 
mastic in chloroform is used, also dammar-varnish, etc. 

The oil of cloves is absorbed by fine blotting- 
paper, and its place is gradually occupied by the 
Canada balsam, which is deposited at the edge of the 
cover-glass. 



42 THE USE OF THE MICROSCOPE. 

Reagents used in the Peocess of Staining. 
The Peinciples oe Staining. — The art of staining 
has become every year more important and indis- 
pensable. Among the results obtained by staining 
the most striking was the discovery of vegetable 
parasites. Weigert, Ehrlich, and Koch have won 
the highest distinction in this field ; the very valu- 
able studies of Ehrlich related substantially to the 
theory of the action of dyes. 

The principle in staining is, therefore, that certain 
elements of tissues, and also of cells, appropriate active- 
ly, or in large quantity, from the solution employed, a 
certain dye, and form with this a combination having 
an intense color, that is more or less permanent. 

The relation of the different substances of the 
human body to the different dyes is naturally a high- 
ly complex one, and we have gradually learned to 
recognize the fact that for almost every tissue-ele- 
ment there is a special dye, or a special method of 
staining, by the action of which it takes a deep char- 
acteristic color, such as distinguishes it from other 
tissue-elements. 

Hence, in many instances staining assumes the im- 
portance of a chemical reaction, by means of which 
any particular structure, that lies concealed among 
other bodies, can be brought easily and conveniently 
into prominence. 

This " elective " action of dyes is of extreme im- 
portance in pathological investigations, as will at 
once be evident; elements which, because of their 
delicate contours, are only to be recognized after very 
careful examination, in the midst of the confused 



REAGENTS. MICRO-CHEMISTRY. 43 

mass of other structures, after "isolated" staining, 
are seen at once, even at a cursory glance, and 
often with a low power, so that we are soon 
compensated for the time spent in the staining pro- 
cess by the gain in convenience and accuracy. The 
process of staining alone frequently discloses to us 
existing differences in tissue- and cell-elements which 
had previously appeared to be perfectly identical. 

It has already been stated that the perfect appli- 
cation of the results of staining was first attained by 
means of the open condenser, or Abbe's illuminating- 
apparatus. The technique of dyeing is usually this : 
A section is transferred from distilled water to a 
dish filled with the staining-solution, with which it 
is entirely covered ; it remains in this for different 
lengths of time, varying from a few minutes to 
twenty-four hours, and is again immersed in distilled 
water, in order to wash away the portions of the 
dye that are adherent to its exterior ; it is then ex- 
amined, either in glycerin directly, or in oil of cloves, 
after dehydration with alcohol. The rationale of the 
staining process in this case is simply that certain 
elements take the dye, while others remain unstained. 
This is called " election." 

In many cases, however, the section which has 
been removed from the staining-solution and washed 
is subjected to further manipulations ; it is again de- 
colorized, that is partially. In this instance there 
has occurred at first a diffuse, even, but unnecessary 
amount of staining ; but during the supplementary 
process of extraction, while certain elements give up 
their staining completely, others, that have a stronger 



44 THE USE OF TEE MICROSCOPE. 

affinity for the dye, retain it. This is called by Ehr- 
licli the principle of maximum staining. This pro- 
cess, which indeed was first employed by the author, 
now plays a prominent part, especially in staining 
with aniline dyes ; alcohol, or acids, serve as the ordi- 
nary extracting-agent. 

It is only in exceptional cases that it is advisable 
to undertake the staining of a section under the 
cover-glass ; the dye is uneven and is confined to 
the edges. Isolated elements, such as cells, can some- 
times be stained under the cover ; however, in this 
case also, the method of coloring dry preparations, 
employed by Koch and Ehrlich, is, as a rule, much 
to be preferred. (Compare the section on " The Ex- 
amination of Fluids.") 

It has long been the custom in embryological and 
zoological investigations to stain organs or animals 
in toto ; alcoholic fluids, especially, have been com- 
pounded which effect simultaneously the hardening, 
as well as staining, of the preparations. The advan- 
tage is that the section can be transferred almost 
directly from the microtome to the slide for examina- 
tion ; aside from the shortness and convenience of the 
process, there is far less danger in this way of injur- 
ing or destroying the section during the different acts 
of staining, washing, etc. But I believe that this 
method is seldom adapted to our ends ; in many, in- 
deed in most cases, we have first the task of giving a 
definite opinion concerning a concrete, practical case, 
and we must therefore always leave ourselves the 
opportunity of applying every available test to the 
specimens under consideration. We should block 



REAGENTS. MICRO-CHEMISTRY. 45 

our own path, as it were, if we carelessly adopted the 
method employed in normal anatomy, since the rela- 
tions are totally different. The normal anatomist, for 
instance, examines as many eyes as he chooses in 
order to decide upon a scientific question ; every nor- 
mal eye is therefore of equal value to him, and for 
every variation in his method he can use a new speci- 
men. But we are often limited to the single speci- 
men before us, from which we must decide upon 
the structural changes that have occurred. To this 
end we must take care to obtain from the object 
(which has been changed as little as possible by 
freezing, hardening in alcohol, etc) a series of sec- 
tions, made through the diseased parts, which we 
may regard as somewhat similar specimens. We 
may then treat these sections according to differ- 
ent methods, and experiment with them, in order to 
study the changes from every side. We can never 
know what surprises we are about to encounter in 
the interior of diseased organs, so that a simple way 
of preparing objects, which disturbs them as little 
as possible before they have been dissected, is the 
best one for us. It is impossible for us before begin- 
ning the examination to adhere to a fixed method, 
but we must reserve for ourselves the possibility of 
taking the longer (since it is the necessary) road, ac- 
cording to the result of examination, with which we 
are not yet thoroughly acquainted. 

We shall, therefore, not describe in this volume 
the process of staining entire organs; if any one 
wishes to employ it, he will obtain the necessary 
hints from Grenacher ("Archiv. fur mic. Anat.," 



46 THE USE OF THE MICROSCOPE. 

Bd. 16), and P. Myer (" Mittheilungen aus der zool. 
Station zu Neapel," Bd. 2, 1880). 

10. Iodine. — This, tlie oldest of tlie staining-ma- 
terials tliat have come into use in microscopic ex- 
aminations, is still used very frequently in tlie form 
of Lugol's solution. Iodine, insoluble in pure water, 
is readily dissolved in a solution of potassium iodide ; 
tlie following mixture is used : 

Pure iodine 1 part. 

Iodide of potassium ... 2 parts. 

Distilled water . . . . 50 " 
This solution can be diluted to any desired extent, if 
necessary. It should be observed that it is difficult 
to preserve the iodine-staining in water and glycerin 
for any length of time ; iodine always forms merely 
a loose combination with organic bodies, and gradu- 
ally evaporates, when the staining vanishes. Even 
when the preparations are carefully mounted, it is 
apt to disappear in a few years at the latest. They 
can not be kept in Canada balsam at all, since the 
color is at once extracted by alcohol. On the other 
hand the iodine-staining seems to be held fast in 
specimens that are placed in a thick solution of 
gum. Albuminous, as well as gelatinous and colloid 
substances, are readily colored yellow by the iodine 
solution ; the cells are usually more deeply stained 
than the interstitial tissue, and the nuclei rather 
more deeply than the protoplasm. The cellular ele- 
ments (as the columns of cells in carcinoma) can 
accordingly be brought out in fresh sections by a 
very rapid and convenient staining process. 

The red blood-corpuscles are stained dark brown 



REAGENTS. MICRO- CHEMISTRY. 47 

by iodine. The following substances give a peculiar 
reaction with, the solution of iodine : 

Glycogen, 

Corpora amylacea, 

Amyloid material. 
Glycogen — In many cartilage-cells, as in those 
of the chorda dorsalis, in the proliferating layer of 
epiphyseal cartilages, even in the normal condition, 
but in an especially striking manner in rickets, as 
well as in enchondromata, an intense wine-red stain- 
ing is obtained with the iodine-solution, which affects 
either the whole, or only a part, of the cell-substance. 
The reaction appears best when the rest of the sub- 
stance is quite faintly stained. The red color de- 
pends upon the glycogen contained within the cells, 
as Neumann and Jaffe have shown ; when not stained 
with iodine the portions that are rich in glycogen 
often present a homogeneous, glistening appearance. 
From a discovery made by Claude Bernard, the same 
condition was long ago recognized in the cells of the 
chorionic villi and other embryonic structures. Boch 
and Hofmann have also, by means of iodine-staining, 
studied the variations in the amount of glycogen 
contained within the liver-cells ; Schiele, a pupil of 
Langhans, also discovered a large amount of glyco- 
gen in normal stratified pavement-epithelium as well 
as in rapidly-growing cancers. 

Ehrlich recently found that if the sections remain 
in watery solutions (it is well known that glycogen 
is insoluble in alcohol) a large part of the glycogen 
is extracted from the cells, and in consequence es- 
capes observation ; he employs, therefore, in order to 



48 THE USE OF THE MICROSCOPE, 

avoid its solution or diffusion, a dilute solution of 
iodine mixed with mucilage, in which the specimens 
are directly examined and preserved. By following 
this method Ehrlich ascertained that in diabetes 
large quantities of glycogen regularly appear in the 
epithelial cells of the renal tubules, especially at the 
boundary -line between the cortex and medulla. 

Corpora Amylacea. — It is well known that starch- 
granules are stained an intense blue color by iodine ; 
they are frequently found in the contents of the 
stomach and intestine, and in the cavity of the 
mouth, and are positively recognized by the iodine- 
reaction ; they are often met with also as chance im- 
purities. The so-called corpora amylacea, which are 
found almost invariably in the nervous system in de- 
generative processes, and in the white substance of 
the brain and cord in elderly subjects, bear a certain 
purely external resemblance to starch-granules ; they 
are to be regarded as products of degeneration of 
the medullary sheath. They assume a deep wine-red 
color with iodine. Certain concretions also, which 
are found occasionally in the lungs, and very often 
in the prostate, are also described as corpora amy- 
lacea. All these transparent or yellowish-brown 
bodies are distinguished by their concentric lamina- 
tion, and by the fact that they stain more or less 
intensely with iodine ; the shade varies from wine- 
red to dark brown. As to their nature and signifi- 
cance little is known ; they have nothing to do with 
starch, and still less with amyloid material. 

Amyloid Material. — Amyloid substance is char- 
acterized by the wine-red color which it forms with 



RE A GENTS. MICE 0- CHEMISTR Y. 49 

iodine ; it is better to use in this case also rather 
weak solutions, having the shade of cognac, so that 
the reaction may take place gradually, in the course 
of several minutes, and be on that account so much 
finer and clearer. 

In many cases of amyloid degeneration the color 
changes to a dark green or blue tint on the ad- 
dition of sulphuric acid ; the sections that have been 
stained not too deeply, but only a clear yellow, are 
placed in a dish containing a one-per-cent. solution of 
the acid, when the reaction occurs at once, or in the 
course of a few minutes. As may be inferred, this 
blue or green staining — by which the degenerated 
portions are brought out from the surrounding tissue 
in a far more striking manner than when iodine- 
staining alone is used — does not occur in all cases of 
amyloid degeneration. The sulphuric acid frequent- 
ly does not alter the shade at all, but merely pro- 
duces a saturated brown color. Furthermore, in 
many cases of amyloid change we find that some of 
the degenerated elements become green or blue when 
iodine and sulphuric acid are added, while others 
only take a dark red staining ; in several cases that 
came under my observation, for example, the arte- 
ries and vasa aff erentia of the kidneys became dark 
red, while the loops of the glomeruli, on the con- 
trary, were dark blue. This colored picture, which 
by reason of its regularity is extremely striking, is 
not very often seen; on the contrary, the amyloid 
arteries are found for the most part to be stained red, 
only a few isolated portions being colored blue. 
These differences in staining may possibly be related 



50 THE USE OF THE MICROSCOPE. 

to the age of the amyloid material, the younger por- 
tions being colored red with iodine and sulphuric 
acid, while the older become blue ; thus I have 
several times observed in the spleen that the arteries 
and capillaries of the enlarged follicles were stained 
blue, while the vessels of the less (or more recently) 
diseased pulp were stained red. Further differences 
between these two varieties of amyloid have not yet 
been established ; when no staining is used, or when 
coloring with aniline dyes is employed, only a uni- 
form appearance is seen, whereas the treatment with 
iodine and sulphuric acid brings out a striking differ- 
ence in color. The iodine reaction has hitherto been 
indispensable for the microscopical diagnosis of amy- 
loid ; amyloid shares its homogeneous, glistening 
character with other colloid and hyaline substances, 
but the latter are only stained a faint yellow by 
iodine. The red color resulting from treatment with 
aniline dyes is not always characteristic, as it ap- 
pears ; for example, we frequently succeed in stain- 
ing urinary casts red with aniline-violet, while they 
are only rendered yellow (or brown if the action is 
stronger) by iodine.* The inference is that even the 
casts that are stained red with aniline do not consist 
of real amyloid substance, but perhaps represent an 
initial stage in the formation of amyloid. 

The chemical character of amyloid material has 
been much studied ; it is known to be a body rich in 
nitrogen, and related to the albuminates. On the 
other hand, the cause of the iodine and iodine-sul- 

* In certain instances casts are also stained reddish -brown with 
iodine. 



REAGENTS. MICRO-CHEMISTRY. 51 

phuric acid reactions, from which amyloid derives its 
highly inappropriate name, is still entirely unknown. 
Nothing is known concerning the red iodine combi- 
nation and the blue bodies into which the latter is 
changed through the action of sulphuric acid. 

It should be noted that cholesterin also stains 
quite dark in a dilute solution of iodine. If a drop 
of strong sulphuric acid is allowed to flow under the 
cover-glass, a beautiful blue color also appears at the 
angles of the tables. 

11. Cakmine. — The introduction of carmine stain- 
ing dates from the year 1858 ; we are indebted for it 
to Harting and Gerlach. 

(a) Ammonia-carmine. — According to Gerlach's 
original formula, the carmine was dissolved in am- 
monia. Add to one part of finely powdered car- 
mine one part of strong liquor ammonise, and from 
fifty to one hundred parts of water. The mixture is 
exposed to the air for twenty-four hours, in order to 
allow the greater part of the ammonia to evaporate, 
and is then filtered. 

The less free ammonia there is in the solution, 
the less injurious is its action upon the tissues ; the 
solution must be frequently renewed, however, since 
it is easily ruined by the formation of mold. 

The carminate of ammonium stains very rapidly 
a large number of the substances found in animal 
bodies. The staining is genuine — that is, it is per- 
manent — especially if the section, after careful wash- 
ing, is placed in dilute acetic acid, for the purpose of 
fixing the color. If the preparation has not been 
washed in exactly the proper manner, it will be com- 



52 THE USE OF TEE MICROSCOPE. 

pletely spoiled by the granular precipitate of carmine 
produced by the acid. 

The following parts are stained : Protoplasm, the 
nuclei of nearly all cells, the fibrillated basement-sub- 
stance of connective tissue, smooth and striated mus- 
cular fibers, the basement-substance of osteoid tissue 
and of decalcified bone, fibrin, the neuroglia of the 
central nervous system, the axis-cylinders of nerves, 
most colloid substances, etc. 

The basement-substance of hyaline cartilage, elas- 
tic tissue, horn, the medullary sheaths of nerves, fat, 
mucus, calcified bone, etc., remain unstained. 

Ammonia-carmine is used principally for examina- 
tions of the nervous system, in order to bring out the 
axis-cylinders. 

Nerve-tissue, as before mentioned, is generally 
hardened in solutions of the chromates ; the longer 
the stay in the latter, so much the slower and 
more difficult is the staining process apt to be, 
so that it often requires several days before the 
proper shade is attained. This drawback is quite 
annoying, especially in the case of very small axis- 
cylinders (in the optic nerve, for instance), that re- 
quire the deepest staining. The staining can be 
rendered more rapid or intense if the solution is 
placed in a compartment warmed to about 50° C. 
(Obersteiner). Under these circumstances the color 
appears in a satisfactory manner, even within an 
hour. 

Heule and Merckel devised another method which 
is worthy of high commendation. Place the section 
first in a solution of chloride of palladium (one to 



REAGENTS. MICRO-CHEMISTRY. 53 

five hundred) for about ten minutes, when it is 
dyed straw-yellow; it is then transferred to the 
carmine-solution, in which it is stained red in a few 
minutes. After careful washing in water, dehydra- 
tion in alcohol, and clearing in oil of cloves, it is 
examined. By this method the medullary sheaths 
are stained yellow, the neuroglia, ganglion-celk, and 
axis-cylinders a deep red. If it is desirable to bring 
out the nuclei also, they can be shown very beauti- 
fully in the carmine section by subsequent staining 
with hematoxylin (compare page 59). Such double 
stainings are to be highly recommended, especially 
for the examination of secondary degeneration of the 
spinal cord, sclerosis, tabes, etc. 

The diffuse red color of the neuroglia in speci- 
mens thus prepared is often troublesome. In order 
to eliminate this, Ranvier places the sections, after 
thev have been stained with carmine, in a mixture of 
one part of formic acid and two parts of alcohol, for 
from five to ten hours ; the axis-cylinders and nuclei 
then remain red, while the neuroglia is deprived of 
color ("Comptes Rendus," November, 1883). 

Aside from this, ammonia - carmine is generally 
used only for the examination of the osseous system. 
Here, too, double staining (with hematoxylin) acts 
very well. The bringing out of the osteoid tissue, 
by means of ammonia-carmine, is of real value, espe- 
cially in the examination of rickets and osteomalacia, 
which is best undertaken with fresh specimens — that 
is, without artificial decalcification. 

Other Carmine Dyes. — A large number of modi- 
fications of carmine-staining have been proposed and 



54 TEE USE OF THE MICROSCOPE. 

recommended ; we mention here only a few of these, 
such as are especially valuable for pathological pur- 
poses. 

(h) Picrocarmine (Schwarz, Ranvier). — The pi- 
crocarmine of the druggists is generally useless ; 
according to Weigert, a small quantity of acetic acid 
is to be added to this in order to form a good stain- 
ing-material ; if a precipitate occurs, it is readily dis- 
solved by a trace of ammonia. 

The author prepares a picrocarmine that stains 
very rapidly according to the following formula : To 
one part of alkaline ammonia-carmine solution (con- 
sisting of one part each of carmine and ammonia, 
and fifty parts of water) add slowly, then drop by 
drop — while stirring constantly — from two to four 
parts of a saturated solution of picric acid, until the 
precipitate that first forms is no longer dissolved by 
stirring; the greater the quantity of ammonia, so 
much more picric acid is it necessary to add. The 
fluid is then filtered, a few drops of phenol are added 
to every one hundred centimetres, in order to pre- 
serve it ? and a cloudiness that appears somewhat later 
is readily dissolved by adding a trace of ammonia. 

This fluid is very useful for most purposes ; it 
produces a double staining within a few minutes. 
The nuclei are all colored deep red, the fibrillated 
substance of the connective-tissue takes a faint 
reddish tinge, while the protoplasmic bodies, on the 
other hand, the smooth and striated muscle-fibers, 
horn, most hyaline and colloid substances, etc., take a 
more or less intense yellow. 

The difference is often still more striking if the 



REAGENTS. MICRO-CHEMISTRY. 55 

sections after being stained are placed for half an 
liour in a disli filled with glycerin containing hydro- 
chloric acid — one part of hydrochloric acid to one 
hundred parts of glycerin ; the picrocarmine solution 
stains with especial rapidity and intensity if it con- 
tains a little free ammonia; in this case, however, 
the carmine dye first preponderates. By treating 
with the acid glycerin the red dye is first removed 
from the protoplasmic and interstitial substances, 
so that the yellow picrine-staining becomes promi- 
nent (Neumann) ; the red color, on the other hand, 
is fixed in the nuclei at the same time. It must be 
observed, moreover, that the red staining of the nuclei 
(in neutral or acid fluids) is permanent, while the 
yellow picrine dye is soon soaked out. In order to 
retain the yellow shade, it is customary to add a 
small amount of picric acid to the water, glycerin and 
alcohol employed, until a slight tinge of yellow ap- 
pears. Then the preparations stained with picrocar- 
mine can be mounted in glycerin just as well as in 
Canada balsam. 

Picrocarmine is a very valuable agent on account 
of the convenient double staining that it causes, 
showing clearly the nuclei on the one hand, and the 
protoplasm of hyaline substance, of horn, smooth 
muscular fiber, etc., on the other. The discovery of 
tubercles in strumous granulations, and in the tissue 
of lupus, has been rendered much easier for the 
author by the employment of this method ; picrocar- 
mine is also to be highly recommended in examina- 
tions of the nervous and osseous systems, and of 
many glands. 



56 THE USE OF THE MICROSCOPE. 

(6) Borax-carmine (Grenadier). — Mix in a porce- 
lain dish and heat to the boiling-point : 

Carmine \ part. 

Borax ..... 2 parts. 

Distilled water . . .100 parts. 
Dilute acetic acid (about five per cent.) is to be add- 
ed drop by drop to the bluish-red fluid, while stirring 
constantly, until the color changes to that of the alka- 
line ammonia-carmine solution ; allow it to stand for 
twenty-four hours, decant and filter, a few drops of 
phenol being added as a preservative. 

A section immersed in this solution is stained 
deeply in a very short time, even in a few minutes ; 
however, the staining is quite diffuse, and is therefore 
useless. On the contrary, the most beautiful stain- 
ing of separate nuclei is obtained, if the deeply stained 
section is placed in a dish full of alcohol that con- 
tains hydrochloric acid, prepared according to this 
formula : 

Hydrochloric acid . . .1 part. 

Alcohol . . . o 70 parts. 

Distilled water . . .30 parts. 
The section at once discharges a part of the dye, and 
is surrounded by a red cloud ; after an interval, vary- 
ing from a few minutes to half an hour, it is washed 
(in water or in alcohol) and is examined in glycerin 
or in oil of cloves. This method gives the most in- 
tense nuclear staining ; in making use of it the action 
of the hydrochloric acid must be taken into account — 
viz., the solution of lime, swelling of fibrin, proto- 
plasm, fibrillated substance, etc. 

(d) Alum-carmine (Grenacher). — A gramme of 



REAGENTS. MICRO-CHEMISTRY. 57 

carmine is heated with one hundred cubic centi- 
metres of a five-per-cent. solution of alum ; boil for 
twenty minutes, and filter after cooling. 

A staining, confined almost exclusively to the nu- 
clei, can be obtained with this solution within from 
five to ten minutes, but it is not quite so intense as in 
the former case. 

(e) Cochineal- Alum Solution (Partsch and Czo- 
kor). — One part of the finest -cochineal (the original 
substance of carmine) and one part of alum are heat- 
ed in one hundred parts of water until about half of 
the solution has boiled away ; a small amount of 
phenol is added and it is filtered. 

The action is quite similar to that of the former 
solution ; I prefer to use it for the simultaneous stain- 
ing of nuclei and axis-cylinders in sections of the cen- 
tral nervous system, after previous hardening in the 
chromates. The staining takes place within twenty- 
four hours, and the nuclei have a different shade 
(deeper violet) than the axis-cylinders. 

(f) Lithium-carmine (Orth). — Two and one- 
half parts of carmine are dissolved in one hundred 
parts of a saturated solution of carbonate of lithium. 
The sections are stained in a few minutes ; after de- 
colorizing in alcohol containing hydrochloric acid 
(comp. under c) a beautiful nuclear staining appears. 

Picro-lithium-carmine is prepared by adding to 
the solution described from two to three parts of a 
saturated solution of picric acid. The color also ap- 
pears rapidly, and presents the advantage of double 
staining, as in the case of picrocarmine. Here, too, 
some of the dye may be removed by means of alco- 



58 THE USE OF THE MICROSCOPE. 

hoi or glycerin, mixed with hydrochloric acid, when 
the contrast becomes greater. 

Since this solution is also quite permanent, it can 
be highly recommended. 

12. H^nrATOXYLix. Weigerfs Method of Staining 
the Central Nervous System. — Staining with hsema- 
toxylin is one of the surest and most excellent ways 
of bringing out the nuclei clearly. 

Hematoxylin crystals readily dissolve in alcohol, 
forming a brown tincture ; if a small portion of this 
tincture be added to a watery solution of alum, a 
bluish fluid results after a few minutes, the staining 
power of which, however, does not reach its full 
height until several days have elapsed ; at the same 
time, or soon after, the dye also begins to be pre- 
cipitated in a granular form, so as to spoil the speci- 
mens. The solution must always be filtered, there- 
fore, just before it is used. In order to obtain a per- 
manent solution, having a constant staining power, 
the f ollowing formula is recommended : 

Hematoxylin, 

Alum, of each ... 2 parts. 

Alcohol, 

Glycerin, 

Distilled water, of each . .100 parts. 
A little acetic acid may afterward be added to the 
mixture, in order to prevent overstaining (Ehrlich). 
It should be observed that the solution only attains 
its full staining capacity eight days after its prepara- 
tion. 

A section immersed in this brown solution is 
stained a similar color in a very short time ; the sec- 



REAGENTS. MICRO-CHEMISTRY. 59' 

tion is washed in distilled water, and in the course of 
a few minutes its color changes to blue. A staining 
confined almost entirely to the nuclei and to (most) 
schizoniycetes is then observed. If other tissue-ele- 
ments have been stained at the same time, these 
should be decolorized with alcohol containing hydro- 
chloric acid ; the staining of the nuclei is well pre- 
served. For many purposes it is desirable to color 
the protoplasmic substance also ; this can be accom- 
plished by subsequent staining with picric acid (satu- 
rated solution), or with eosin (see under Eosin). The 
specimens stained in hematoxylin gradually lose 
their color when kept in glycerin, and hence are 
best preserved in Canada balsam. 

Weigert* has taught us quite recently a most 
valuable use of hematoxylin staining, in connection 
with the central nervous system, whereby it is possi- 
ble to exhibit, in a very elegant manner, the fine 
medullated nerve-fibers, which could previously be 
shown only with the greatest difficulty. The method 
is a peculiar one, and is somewhat complicated. The 
portions of the central nervous system are hardened 
in Muller's or Erlitzski's fluid (compare page 34), 
and are then transferred to alcohol without previous 
soaking ; the sections, before they are dyed, must also 
be placed, not in water, but only in alcohol. The 
staining-solution consists of one part of hematoxylin, 
ten parts of alcohol, and ninety parts of water ; the 
mixture is boiled, and allowed to stand for a few days. 
The sections stain best if they are placed for one or 
two hours in an incubator at a temperature of 40° C, 

* " Fortscbr. d. Med.," Bd. ii, S. 190. 



60 THE USE OF TEE MICROSCOPE. 

and are then washed in water. The deep black sec- 
tions, which are greatly over-stained, are then almost 
entirely decolorized by immersion for half an hour 
or an hour in a mixture of two and one half parts of 
ferrocyanide of potassium, two parts of borax, and 
one hundred parts of water, until the gray matter 
appears yellow ; the white substance remains black. 
The sections are then thoroughly washed in water 
(ferrocyanide of potassium is precipitated by alcohol), 
and placed successively in alcohol, xylol and Canada 
balsam. 

With this staining the nerve-medulla comes out 
very sharply, by reason of its dark color, w^hile the 
axis-cylinders, ganglia, cells, nuclei, etc., remain nearly 
colorless. 

By employing Weigert's method, I have succeed- 
ed in demonstrating positively the change in the gray 
substance in tabes, which has long been asserted, 
but which has hitherto been sought for in vain — 
namely, the disappearance of the network of delicate 
nerve-fibers in the interior of the columns of Clarke. 
This discovery is of great theoretical importance. I 
have described the method minutely, since it is quite 
possible that other changes will be found in the cere- 
bral cortex in progressive paralysis, secondary de- 
generations, retinal affections, etc. The specimens 
obtained in this way are extremely useful ; the nuclei 
may easily be stained subsequently, by means of alum- 
carmine. 

13. Eosen t . — Eosin forms a fluorescent solution, 
giving a rose-red color with transmitted, and a green- 
ish color with direct light, which, even with a strength 



REAGENTS. MICRO-CHEMISTRY. 61 

of one to one thousand, imparts a deep rose-red stain- 
ing to sections within a few minutes. The staining is 
generally very diffuse, affecting the most diverse sub- 
stances ; even the red blood-corpuscles assume an in- 
tense rose-red shade, which is still deeper in sections 
made from alcoholic specimens, provided that the 
chromates have been used for hardening. The color 
is removed by absolute alcohol, at first very rapid- 
ly, then more slowly, so that, by the careful use of 
this agent, any desired shade can be produced. 

A pure eosin-staining is very seldom of advantage 
(concerning the "eosinophil" cells, see below), al- 
though eosin is very often used for double staining, 
in connection with the coloring of nuclei. For this 
purpose the nuclei are, to bring out the contrast, best 
dyed blue, with gentian or methyl- violet (q. v.) ; or 
with hematoxylin. 

A mixture of hematoxylin and eosin can be pre-^\ 
pared, according to Renaut's suggestion, in which the 
double staining is effected at once. It is only neces- 
sary to add half a part of eosin to the solution of 
hematoxylin, already mentioned, in order to obtain 
such a mixture. The sections are usually too strong- 
ly stained with eosin at first ; but, after a short stay 
in alcohol, the proper shade generally appears, where- 
upon it is better to examine the section in oil of 
cloves. 

This method is, in a great many cases, the most con- 
venient and the best for staining prepared sections. 
The nuclei of lymphoid cells generally appear to be 
most deeply colored, next those of the capillaries, of 
the other endothelia, and of connective tissue ; while 



62 THE USE OF THE MICROSCOPE. 

the nuclei of epithelial cells, etc., are less affected. 
Furthermore, differences appear in the depth of 
staining of the protoplasm, such as render pos- 
sible the recognition of single elements, even with 
a low power. The parietal cells of the gastric glands, 
the giant cells of tubercle, etc., take a particularly 
deep color. 

14. Aniline-Black (Nigbosin). Aniline-Blue. 
— These two dyes have a very similar action : they 
are used for staining axis-cylinders in sections of the 
nervous system. The section is immersed in a solution 
of about one per cent., in which it is deeply stained 
in a few minutes, and it is then washed in alcohol, 
and is again almost entirely decolorized. When the 
proper shade has been reached (which can easily be 
ascertained) the section is placed in oil of cloves 
or Canada balsam; the axis-cylinders and ganglion- 
cells take a very convenient blue or black tint, 
which is less marked in the neuroglia. They may 
be used also for staining protoplasm; quite weak 
solutions, for example, cause a very dark and charac- 
teristic coloring of the parietal cells (formerly called 
" peptic cells ") of the gastric glands. 

15. The Basic Aniline Dyes, which stain Nu- 
clei. — The following basic aniline dyes, which stain 
nuclei, are the ones principally used : 

Vesuvin (Bismarck-brown). 
Fuchsin. 

Gentian and methyl-violet. 

Methyl-blue ; also dahlia, magdala, methyl-green, 
etc. 

These different substances possess nearly the 



REAGENTS. MICRO-CHEMISTRY. 63 

same peculiarities with, regard to their action upon 
tissues, so that they may be considered together. 
They are all easily soluble in alcohol and in water, 
but their alcoholic solutions are only rarely used ; we 
almost always employ the watery. It is better to 
keep on hand a concentrated watery solution which 
contains an excess of dye, and to filter off the neces- 
sary quantity from this fluid just before using. It is 
often convenient to have, instead of this quite opaque, 
deeply colored solution, a thinner one that acts less 
powerfully (about one to one hundred), to which, on 
account of its strength, a tenth part of its volume of 
alcohol may be added. 

Staining of Nuclei. Cells without Nuclei. — If 
a section be placed in this solution it is stained an 
extremely dark color in a short time — within a few 
minutes. If it is washed in water and examined, we 
find a color that is almost completely diffused, is uni- 
form, and is consequently useless. The advantages 
of the staining first appear after the action of the 
alcohol has taken place ; the section when placed in 
a dish of alcohol discharges a large amount of dye, 
and is surrounded by a colored cloud ; when re-exam- 
ined after a few minutes, a beautiful, distinct staining 
of the nuclei is observed, and those of lymphoid cells, 
as well as those of connective-tissue and endothelial 
cells, have usually a much darker tinge than the nu- 
clei of epithelial cells. The other substances are all 
nearly colorless, with some few exceptions to be men- 
tioned shortly. The process is, therefore, soon at an 
end, the section being placed for a few minutes in 
the staining solution, and then for a few minutes in 

4 



64: THE USE OF THE MICROSCOPE. 

alcohol ; from the latter it is either transferred to oil 
of cloves, in which the unstained portions become 
almost entirely cleared up, and is finally mounted in 
Canada balsam; or it is again placed in distilled 
water and is examined in glycerin. It should be re- 
marked in this connection that the colors are well 
preserved in balsam-preparations, while in glycerin- 
specimens the nuclear staining is only retained when 
Bismarck-brown or vesuvin is used, while methyl- vio- 
let staining gradually disappears in glycerin (Wei- 
gert). For this reason Bismarck-brown is to be very 
highly recommended for most purposes, especially for 
the staining of fresh sections ; these do not cause pre- 
cipitates in solutions of Bismarck-brown, while they 
often occasion very troublesome granular deposits in 
a solution of methyl- violet. 

By means of the distinct, separate staining of 
nuclei wirich can be produced by hematoxylin, picro- 
carmine, borax-carmine, lithium -carmine, etc., as well 
as by the aniline dyes already mentioned, "Weigert 
was led to the important discovery that in a large 
number of pathological processes the cell-nuclei are — 
either invariably, or at least in certain types of cells — 
destroyed, a fact that had previously remained almost 
unknown. The absence of nuclei in the cells of the 
deep layers of the epidermis in small-pox was first dis- 
covered, then in diphtheria, later in various organs in 
the neighborhood of colonies of micrococci, in the 
looped tubules of the kidney in chromic-acid poison- 
ing, in renal infarctions, in cheesy degeneration, etc. 
It was soon proved that the nuclei are absent in ne- 
crosed cells which have remained exposed for some 



REAGENTS. MICRO-CHEMISTRY. 65 

time after death to a current (although diminished) of 
nutrient fluid ; in many cases the protoplasm of the 
cells assumes simultaneously a glistening, homogene- 
ous appearance, and these are the cases to which 
Cohnheim has applied the term " coagulation-necro- 
sis." This expression has since been greatly misap- 
plied ; in my opinion it should be limited to those 
cases in which coagulation, as well as necrosis, has 
been demonstrated, or at least is probable ; at all 
events, every cell in which a distinct nuclear staining 
does not occur should not be regarded as the seat of 
" coagulation-necrosis." It should be observed that 
when aniline dyes are used the nuclei of epithelial 
cells that are quite normal may, under certain circum- 
stances, appear perfectly colorless if they are stained 
in an acid solution, or if dilute acetic acid as well as 
alcohol is used for decolorizing. Besides, it has not 
been positively proved that every cell in which a 
nucleus can not be demonstrated is therefore to be 
at once regarded as necrosed ; it is always well in 
such doubtful cases of cells with non-staining nuclei, 
not to refer at once to coagulation-necrosis. Before 
the nucleus entirely disappears there are often found 
in its stead small, deeply-stained granules, which 
may be regarded as the products of disintegration 
of the nucleus, but which are often mistaken for 
micrococci by the unskilled ; they are to be at once 
distinguished from these organisms by their very 
variable size. 

The following bodies are stained by the above 
process, in addition to the nuclei : 

1. The basement-substance of hyaline cartilage. 



66 THE USE OF THE MICROSCOPE. 

2. The mucous substances, as well as mucus in 
the glands. 

3. Most of tlie micrococcus- and bacillus-forms; 
further mention will soon be made of these. 

4. Certain protoplasmic granules, as the proto- 
plasm of the so-called " food-cells." 

Food - Cells (Mastzellen). — The " f ood-cells," 
most thoroughly studied by Bhrlich, are somewhat 
bullet-shaped (sometimes flat and fusiform) bodies, 
which are about twice as large as lymphoid cells; 
they consist of a rather coarsely granular proto- 
plasm, the granules of which are deeply stained 
with basic aniline dyes according to the method 
already described. The granules take a reddish 
color when the violet dyes are used; but the nu- 
cleus always remains unstained, and appears as a 
bright spot in the midst of the deeply stained pro- 
toplasmic particles. They are widely distributed 
throughout connective tissue, and are especially 
abundant in mucous membranes, in submucous and 
also in inter-muscular tissue, in serous membranes, 
etc., generally in the vicinity of vessels, but almost 
always singly. Their physiological and pathologi- 
cal significance is still very little known ; it has not 
been proved that they bear any relation to nutri- 
tion. They are found in large numbers in slowly 
forming granulation, or connective-tissue growths, as 
in elephantiasis and in the neighborhood of tumors. 
They have also been found in leucaemic blood, but are 
absent from normal human blood. 

The Staining of Amyloid with Violet Ani- 
line Dyes. — The violet basic aniline dyes, methyl- 



REAGENTS. MICRO-CHEMISTRY. G7 

violet and gentian- violet, show an interesting reaction 
with amyloid substance, as was discovered almost 
simultaneously by Heschl, Juergens, and Cornil ; 
they stain this a deep red, while the nuclei are 
stained blue. The red color of the amyloid is pre- 
served in dilute acids ; in alcohol, on the contrary, it 
is at once discharged, while the similar reddish stain- 
ing of the food-cells is retained in alcohol. We can 
not, therefore, decolorize specimens thus stained in 
alcohol in the usual manner, but we use for this pur- 
pose a dilute acid, such as acetic (one to one hun- 
dred) ; they must not be stained too deeply at the 
outset, hence a rather dilute solution (about one to 
one thousand) is used, which gives a sufficient color 
within a few minutes. The sections are examined 
and preserved in glycerin, in which, however, the 
nuclear staining is lost after a little while. 

The difference in color between the red amyloid 
and the other blue portions is very striking ; at the 
same time there is also in well-prepared specimens 
a distinct blue staining of the nuclei. In conse- 
quence, this process possesses great advantages over 
the dyeing of amyloid with iodine, or iodine and sul- 
phuric acid, which was formerly the only one known. 
It is well known that by the systematic use of this 
method Eberth has learned the important fact that 
the cellular elements — for example, those of the liver, 
kidneys, and spleen — as well as the smooth muscular 
fibers, of the vessels, never undergo amyloid degenera- 
tion, but that this is always confined to the interstitial 
substance and homogeneous membranes. The aniline 
method is decidedly superior for the decision of such 



68 THE USE OF THE MICROSCOPE. 

questions ; on the contrary, the caution must be given 
that every red staining with methyl-violet does not 
necessarily denote actual amyloid degeneration ; other 
hyaline bodies also, which are perhaps related to amy- 
loid material, such as certain urinary casts, show the 
reaction, but they should not on that account be re- 
garded as amyloid. The two kinds of amyloid which 
have been distinguished, by means of their different 
reactions with iodine and sulphuric acid, conduct 
themselves in a similar manner toward the aniline 
dyes. 

Curschman has recently recommended methyl- 
green for amyloid staining ; the amyloid portions be- 
come violet, while the normal tissue, especially the 
nuclei, are green. The difference is certainly very 
striking ; however, the violet color of the amyloid 
only results from a chance mixture of methyl- violet 
with the impure commercial methyl-green. As a 
rule, the use of impure drugs is not to be recom- 
mended for our purposes; it is better to adhere to 
substances that are exactly defined chemically, and 
are in the purest condition. 

Identification and Staining of Schizo]viycetes. 
— The staining of schizomycetes has borne an essen- 
tial part in the important discoveries which have 
been made during the last few years in the province 
of infectious diseases ; there is, therefore, every reason 
why this process should be exactly described. 

(a) The Identification of Schizomycetes when uti~ 
stained. — In order to show schizomycetes when they 
are not stained, we generally take advantage of a 
peculiarity possessed by them, namely, their resist- 



REAGENTS. MICRO- CHEMISTRY. 69 

ance to acids and alkalies. A section made from a 
fresh or alcoholic * specimen is rendered nearly trans- 
parent by strong acetic acid, or a dilute solution of 
potassium or sodium (about two per cent.). Among 
the few elements which resist this treatment the 
schizomycetes are at once recognized, by the char- 
acteristic form of the individual organisms. This 
applies particularly to the bacilli ; in their case it is 
quite possible that minute crystals may give rise to 
error. In fact, the bacilli of typhoid fever and tuber- 
culosis, for example, may thus be demonstrated very 
well in organs ; it is better to employ, for this pur- 
pose, specimens which have been kept only a short 
time in alcohol, since, as a rule, the sections after- 
ward cease to be perfectly transparent. Klebs and 
Baumgarten have laid particular stress upon this 
point ; however, it is nearly always possible, even in 
the examination of old alcoholic preparations, to clear 
up the sections sufficiently, by warming them for a 
short time, under the cover-glass, with liquor potassse 
or acetic acid, until bubbles begin to form. 

Secondly, by the characteristic union or grouping 
of the individual organisms. This consists in the for- 
mation either of chains (diplococci, streptococci, strep- 
to-bacteria, etc.), or of the so-called colonies (gliacoc- 
cus). It is possible only in very exceptional cases to 
mistake these for inorganic precipitates ; they may be 
at once distinguished from very fine fat-drops by the 

* Hardening in chromic acid, Muller's fluid, etc., is not suitable for 
the examination of micro-organisms, since by the action of chromium 
numbers of dark granules are formed in the tissues, which are very 
difficult to clear up. 



70 THE USE OF TEE MICROSCOPE. 

fact that they resist the process of extraction with 
ether and chloroform. 

It is easy to recognize the micrococcus-chains and 
groups by the peculiar glistening appearance of the 
separate granules, or by their color (brownish), which, 
moreover, is only observed in certain varieties, when 
several granules are placed one above the other, also 
by the nearly uniform character of the separate 
bodies, as well as by their clear, sharply-limited out- 
lines under a high power. We are perfectly certain 
that we have to do with organisms, if we succeed in 
proving the fact that they grow. This it is possi- 
ble to do if they develop in the interior of vessels, 
because in growing they distend the lumina un- 
equally ; since the rapidity of their growth varies at 
different points, varicose swellings of the vessels re- 
sult. This occurs very often in the blood-vessels 
(capillaries and small veins) in cases of metastatic 
pyaemia, ulcerative endocarditis, etc. ; the author 
found a similar varicose injection of schizomycetes in 
the lymph-vessels in acute croupous pneumonia. 
These forms of "capillary emboli," resulting from 
unequal filling of the capillaries with granular mate- 
rial, were formerly observed, and it was then known 
that such emboli cause inflammatory foci; but this 
granular matter was described as "detritus" until 
Recklinghausen, and very soon after Klebs, Waldeyer, 
and others, led especially by the varicose form of the 
injection, made the important discovery that the 
" granular detritus " represents living parasitic organ- 
isms, or micrococci. For only a substance which at- 
tains a peculiar capacity for growth can cause such 



REAGENTS. MICRO- CHEMISTRY. 71 

an irregular, nodular form of vascular injection. 
After this identification lias once been assured, it is 
of course not necessary to show signs of growth in 
every single case in order to establish the diagnosis 
of a micrococcus-colony. If we find, in a section 
made either from a fresh organ, or from one that has 
been hardened in alcohol, groups or chains of small 
granules, which are of nearly equal size, which resist 
treatment with alcohol and ether, as well as the ener- 
getic action of concentrated acetic acid and alkalies, 
even under the influence of heat, we are justified in 
calling these granules organisms. 

In all important, or at all doubtful, cases we shall 
of course prefer the staining-reaction, to be presently 
described; staining is naturally of great advan- 
tage also for finding schizomycetes in sections. I 
shall not discuss here the methods of cultivation, 
but refer to Koch's classical description in the com- 
munications of the Imperial Board of Health for 
1881. 

(b) Staining of Micrococci, etc. — Schizomycetes 
generally act toward dyes in a manner very similar to 
the substance of nuclei, from which, however, they 
are to be clearly distinguished by their resistance to 
alkalies, in which nuclei are at once dissolved. The 
methods of staining nuclei are therefore suitable for 
staining most schizomycetes also, and since we have 
here to deal with very minute bodies, we endeavor 
to obtain the deepest color possible; dyeing with 
hematoxylin and the nucleus-staining carmines is 
less adapted for this purpose than the basic aniline 
dyes (Weigert). We accordingly use very concen- 



72 THE USE OF TEE MICROSCOPE. 

trated watery solutions of the latter substances, since 
the addition of a large quantity of alcohol injures 
their staining power ; the intensity of the color may 
be still further increased, according to Koch, if the 
staining is carried on in the incubator at a tempera- 
ture of about 50° C. The process requires from a 
few minutes to half an hour ; the subsequent decolor- 
ization in alcohol (the alcohol must be pure, and above 
all perfectly free from acid) must not be too pro- 
longed, since the color of the schizomycetes also is 
sometimes gradually removed by alcohol ; the speci- 
men is always mounted in oil of cloves, or in Canada 
balsam, and is examined by the aid of Abbe's illumi- 
nating apparatus, with the open condenser. It is 
generally of small importance which of the before- 
mentioned dyes is used ; preparations stained with 
vesuvin or Bismarck-brown can be photographed, 
while the blue or red staining with gentian-violet or 
fuchsin usually presents the most striking and vivid 
pictures. 

The stained micrococci (bacilli are unmistakable) 
might be considered either as the remains of nuclei, 
which, however, are generally to be distinguished at 
once by their variable size, and by their location at 
the site of the original nucleus, or as the granules 
of food-cells ; but the latter are always easily recog- 
nized as such, by reason of their grouping around the 
central unstained nucleus. Many apparently insuper- 
able obstacles to the recognition of these objects cer- 
tainly do confront him who is not accustomed to ac- 
curacy in his work and observations. I have often 
noticed that an observer was inclined to regard as 



RE A GENTS. MICRO- CHEMIST R Y. 73 

micrococci irregular, stained mucus-coagula, or other 
purely accidental objects. Others allow their sec- 
tions to lie for days in impure water, instead of in 
alcohol. Numbers of schizomycetes then collect both 
on the surfaces, and at the edges of the section, so 
that, when the latter is subsequently stained, per- 
plexities arise as to their meaning. There are also 
a number of minds which either never attain the ca- 
pacity for the more delicate histological examinations, 
or succeed only after long and painful training. 

Are all parasitic micro-organisms stained in this 
way ? No ; just as there is no one general method 
of histological examinations, so we can not expect to 
discover any single method of demonstrating micro- 
organisms. Micrococci and, so far as we know at 
present, all varieties of them, are stained in the 
manner described. Only the limitation must be im- 
posed that they generally lose their power of ab- 
sorbing or retaining coloring - matter after death. 
There are frequently found in the interior of organs, 
beside the deeply stained groups of organisms, much 
paler, even quite colorless, collections of granules, 
which may, with the greatest probability, be called 
dead micrococci. In the micrococcus - chains, also, 
differences in the intensity of the color are observed 
in the individual members of the chain, which are to 
be explained in the same way. 

It is always possible that further study will re- 
veal differences in the staining power of the various 
species of micrococci ; as yet, however, nothing is 
known on this subject. 

The micrococci of malignant endocarditis, of py- 



74 THE USE OF THE MICROSCOPE, 

senna, erysipelas, gonorrhoea, etc., act in qnite a uni- 
form manner with the methods wliich we employ. 

It is well known that the micrococci of pneu- 
monia are frequently distinguished by a peculiar cap- 
sule ; this stains only slightly with gentian - violet 
and fuchsin, while the coccus itself assumes a very 
dark sliade. The capsule and the coccus stain almost 
uniformly with. Bismarck-brown and methyl-blue. 

The spirilli of recurrent fever occupy a peculiar 
position. Most observers have not succeeded in iden- 
tifying these in situ in the interior of organs. The 
spirilli are known to be differently constituted from 
most of the other sckizomycetes, since they are quick- 
ly destroyed by acids and alkalies, and even by dis- 
tilled water. They also resemble protoplasm in their 
behavior, rather than the substance of nuclei. They 
can not, therefore, as a rule, be colored by the ordi- 
nary process of nucleus-staining. Koch alone, the 
greatest master in this art, has succeeded in staining 
them, even in the interior of organs, by means of brown 
aniline dyes, and has photographed them. Even he 
declares that the identification of spirilli in hardened 
organs is a difficult task. 

Bacilli act differently ; the forms that usually ap- 
pear during the decomposition of the body, as well 
as the bacilli of splenic fever, stain deeply. The 
staining power of typhus - bacilli was at first ques- 
tioned, but wrongly; they only dye somewhat less 
intensely than the others, and even this slight differ- 
ence completely disappears if the process is carried 
on in a warm chamber. On the other hand, small 
unstained spots, or apparent gaps, partly circular, 



REAGENTS. MICRO-CHEMISTRY. 75 

partly elliptical, are found in the interior of the ty- 
phus-bacilli, which include about half the breadth of 
the bacillus, and sometimes more. These may possi- 
bly be the spore of the bacilli, or they may be " vac- 
uoles.' ' According to Goffky, another kind of spore- 
formation takes place in typhus-bacilli, when culti- 
vated at the temperature of the body — namely, spores 
of the same breadth as the bacillus, attached together 
end for end. 

The bacilli of leprosy stain with gentian-violet, 
methyl-violet, and fuchsin, but not with Bismarck- 
brown. In them, also, are found colorless spots simi- 
lar to those in the bacilli of typhus fever. 

(6) Gram's Method. — While, by the method 
which has been described (which until recently was 
the only known way, of staining schizomycetes), the 
parasites were colored in common with the nuclei, 
Dr. Gram, of Copenhagen, my esteemed friend and 
co -laborer, has lately succeeded in discovering a 
process of separate staining of schizomycetes, which 
may be regarded in most cases as the best, and even 
as the ideal method.* 

In many instances, the deeply stained nuclei con- 
ceal the schizomycetes, which are always much smaller, 
and are, therefore, only slightly dyed. For this reason 
it is necessary, in the case of tissues that are rich in 
nuclei, such as the liver, spleen, lymph-glands, pneu- 
monic lungs, granulation-tissue, etc., to make use of 
the old process of Weigert, and to employ very thin 
sections. Even then it is often quite difficult to see 
and to demonstrate the delicate objects. Gram's 

* Gram, "Fortschritt d. Med.," 1884, S. 185. 



76 THE USE OF THE MICROSCOPE. 

method effects a separate staining of the schizomy- 
cetes. While everything else remains perfectly color- 
less, they, on the contrary, are dyed an intense blue, 
so that almost every individual in the section must at 
once strike the eye of the beholder. 

The method is frequently purely empirical. Ehr- 
lich's solution of gentian - violet in aniline water is 
used for staining. Some finely - powdered gentian- 
violet is shaken into a glass flask, to the depth of 
two finger-breadths ; upon this pour aniline- water — 
that is, a saturated solution of aniline (a yellowish, 
oily fluid), which is at once formed by stirring four 
parts of aniline in one hundred parts of distilled 
water. The excess of aniline, which is partly pre- 
cipitated and partly suspended in the water, so as to 
form a light cloud, is separated by filtration. The 
aniline- water, which is now perfectly clear, is poured 
upon the dry gentian-violet, and, in the course of 
twenty-four hours, after violent shaking, a saturated 
staining-solution is produced. We accomplish almost 
the same end by pouring about five parts of a sat- 
urated alcoholic solution of gentian-violet into one 
hundred parts of aniline - water. If the bottle is 
loosely stoppered with a funnel lined with filter- 
paper, the necessary quantity of the solution can be 
filtered off into a dish without further ceremony. 
The same filter can be frequently used, and the solu- 
tion is preserved unchanged for several weeks, and 
even months. The section of the alcoholic specimen 
which is to be examined (or the cover-glass to which 
the dried preparation is adherent) is placed in this 
solution for a few minutes ; the section must have 



EE AGENTS. MICRO-CHEMISTRY. 77 

previously been immersed in pure alcohol, not in 
water nor in dilute alcohol. The very deeply stained 
sections are then transferred to a dilute solution of 
iodide of potassium (iodine one part, iodide of potas- 
sium two parts, distilled water one hundred parts), 
when a dirty precipitate is formed. They are left 
in this for from one to three minutes, and are then 
removed to absolute alcohol. The color is soon com- 
pletely discharged in the latter, and the alcohol is 
colored purple-red, while the sections become almost 
colorless. They are cleared in oil of cloves, and are 
mounted in xylol, Canada balsam, glycerin, or glycer- 
in-cement. 

"While all of the tissue-elements appear perfectly 
colorless (sometimes a dull, bluish shade remains in 
the nuclei), the schizomycetes stand out as dark blue, 
almost bluish - black, granules or rods, threads, etc. 
It is at once evident that the micro-organisms are 
observed far more clearly, and in greater numbers, 
than by the former method of staining the nuclei. It 
is possible that we shall succeed in this way in dis- 
covering the probable parasites of those infectious 
diseases (as syphilis, typhus, dysentery, etc.) the pa- 
thogeny of which has hitherto been almost, or entire- 
ly unknown. 

By the subsequent use of Bismarck-brown a very 
beautiful double staining may be produced, the schi- 
zomycetes remaining dark blue, while the nuclei be- 
come yellow or brown. In this way the relative 
positions of the parasites in the tissues appear more 
clearly, so that we learn whether they are situated 
within, or outside of, the cells. 



78 THE USE OF THE MICROSCOPE. 

It should be observed also that with this process 
the typhus-bacilli appear colorless, or are decolorized 
like nuclei, so that they are distinguished in this way 
from most of the bacillus forms. In many cases of 
pneumonia the cocci are almost entirely decolorized 
by the use of the iodine method ; in most cases, how- 
ever, they appear in enormous numbers, far more 
numerous than one would believe from using the old 
process of Weigert ; a comparison of both methods 
applied to sections of the same series is very instruct- 
ive. It must always be observed that there are 
cases in which the iodine method does not bring out 
the schizomycetes clearly, while they can be shown 
in another way. 

(d) The Staining of Tubercle-Bacilli. — The pecul- 
iar relation of the tubercle-bacilli to aniline staining 
is of great importance. After efforts had long been 
made in vain to demonstrate anatomically the excit- 
ing cause of tuberculosis — the statements for and 
against the discovery of micrococci in tubercles 
were not well supported — Koch again succeeded, 
by the use of a modified staining process, in demon- 
strating the constant presence of a specific bacillus 
in the products of tuberculosis.* Since this concerns 
one of the most important discoveries of recent medi- 
cine, I desire to introduce here the original method 
of Koch, although it has been superseded by Ehr- 
lich's modification (which was published soon after), 
and is practically only seldom employed. Koch's di- 
rections were as follows : The section (or the dry 

*Koch, a Die iEtiologie der Tuberculose." "Berlin, klin. Woch- 
enschr.," 1882, No. 15. 



REAGEXTS. MICRO-CHEMISTRY, 79 

preparation) is immersed for twenty-four hours in a 
mixture consisting of two hundred parts of distilled 
water, one part of a concentrated alcoholic solution of 
methyl-blue, and one fifth of one part of liquor potassse 
(ten-per-cent. solution). It is now stained dark blue, 
and is next placed for fifteen minutes in a concentrated 
watery solution of vesuvin. It is then washed in dis- 
tilled water till the blue color has disappeared and a 
more or less dark brown shade remains ; it is next de- 
hydrated in alcohol, is cleared in oil of cloves, and is 
examined under a homogeneous lens, w r ith the open 
condenser. The nuclei, as well as most of the varie- 
ties of micrococci, then appear of a brown color, as if 
they had only been exposed to the vesuvin solution ; 
the tubercle-bacilli, on the contrary, are stained a 
deep blue. Koch was of the opinion that this depend- 
ed upon the alkaline reaction of the staining solu- 
tion, since the bacilli were never stained in acid or 
neutral dyes ; the neutral solution of another dye 
then displaced the first staining everywhere except in 
the tubercle-bacilli, which retained the original color. 
There is also a specific reaction which affects the 
tubercle-bacilli only, in contrast with other schizomy- 
cetes ; the bacilli of leprosy, according to Koch, be- 
have in a similar manner, and they also retain the 
blue staining. The latter, however, are also dyed by 
employing the simple nuclear staining, with which the 
tubercle-bacilli stain only a little, if at all (see above). 
The modification which was soon after introduced by 
Ehrlich depends upon the following principles: In 
the first place, in order to render the staining solution 
alkaline, he does not use liquor potass^B, but another 



80 THE USE OF THE MICROSCOPE. 

base, namely, aniline, a yellowish oily fluid, a satu- 
rated watery solution of which is able to dissolve 
far more of the dye than the dilute solution of pot- 
ash. Then he employs strong mineral acids for de- 
colorizing. He proceeds upon the principle that the 
tubercle-bacilli, which according to Koch remain 
colorless in neutral and acid dyes, but which are, on 
the contrary, stained by alkaline solutions of the 
same dyes, are surrounded by a cloud that is only 
permeable for fluids having an alkaline reaction. If, 
therefore, the staining has taken place in the alkaline 
solution, the nuclei, protoplasm, basement substance, 
etc., as well as the bacilli, are colored, so that the 
latter remain concealed. If, however, the specimen is 
placed in acid, the color will be removed from all the 
other elements, and in time even from the other mi- 
cro-organisms that may be present in the preparation, 
since the acid bears a very close relationship to the 
dye. But the acid can not affect the bacilli, because 
their supposed capsules are impermeable to acids; 
the bacilli, therefore, remain as the only colored bod- 
ies in the midst of the otherwise perfectly colorless 
specimen, and thus stand out in the most striking 
manner. 

Whether this hypothesis of the capsules of bacilli, 
impermeable to acid and neutral, but permeable to 
alkaline solutions, is right or not, the future must de- 
cide ; at any rate, it explains most of the phenomena 
hitherto known, and has led to the discovery of a 
method through which the tubercle-bacilli, and, in- 
deed, these only, are deeply stained in tissues and 
fluids. 



REAGENTS. MICRO-CHEMISTRY. 81 

The correctness of Ehrlich's hypothesis has been 
called in question by Ziehl ; the latter also succeeded 
in staining tubercle-bacilli by adding phenol (a sub- 
stance which generally acts like an acid) to ani- 
line dyes. Furthermore, it was stated by Lichtheim,* 
Giaconii, and others that simple aqueous solutions of 
gentian-violet and fuchsin are capable, if sufficient 
time be allowed (the process occurs rapidly on the 
application of heat) of staining the tubercle-bacilli, 
though not so deeply as by Ehrlich's method. A 
large number of new methods of coloring bacilli 
have been published, but these are all, so far as they 
can be relied upon, only more or less important modi- 
fications of Ehrlich's. Since the latter, so far as is at 
present known, is perfectly reliable and has not been 
surpassed by any others, I shall confine myself to 
an accurate description of this process, which was 
at once accepted, even by Koch himself ; f I my- 
self have never had any reason to abandon it. An 
account of other so-called " methods," such as have 
been published, would be very uninteresting and 
without any practical value. 

The following elementary principles should be 
remembered : While most of the other schizomycetes 
are rapidly and deeply stained by aqueous solutions 
of gentian-violet, fuchsin, Bismarck-brown, etc., this 
does not occur in the case of tubercle-bacilli ; the con- 
tinuous action of the dye is necessary, the specimen 
being kept in the warm chamber. Success in stain- 
ing tubercle-bacilli deeply has not generally been at- 

* " Fortschr. d. Med.," Bd. 1. 

t " MittheiluDgen aus deui k. Gesundheitsamt," Bd. 2, 1884. 



82 THE USE OF THE MICROSCOPE. 

tained by the use of Bismarck-brown ; gentian- violet, 
or fuchsin, is used. Aniline-water — that is, a satu- 
rated solution of aniline in water — is employed as a 
solvent. When the staining has taken place, the 
specimen is treated with mineral acids, such as hydro- 
chloric, nitric, etc., in watery or alcoholic solution. 
Those bacilli which retain their color after treatment 
with strong mineral acids are to be regarded as 
tubercle-bacilli. Their peculiar property of retaining 
their color in strong acids is perfectly characteristic 
of them; the diagnosis of tubercle-bacilli should 
only be regarded as confirmed if the staining has 
been retained after the use of acids. I hold that 
it is by no means allowable to omit the treatment 
with acids, simply on the ground of convenience. In 
properly prepared specimens all the other tissue-ele- 
ments must be perfectly colorless, even the other 
varieties of schizomycetes which may be present, so 
that by completely extinguishing the structural image 
— that is, with the full Abbe's illumination — the 
bacilli remain quite alone in the field. In order 
to bring the tubercle-bacilli more easily into view, 
as well as to render clear their relative positions 
in the tissues, the nuclei may also be stained, in 
the manner described, with a color such as will offer 
the strongest possible contrast. The process is car- 
ried out in the following manner : The preparation 
of the staining fluid, gentian-violet in aniline-water, 
has already been accurately described (page 76). 
Fuchsin, or various other aniline colors (such as mag- 
dala, dahlia, methyl-violet, magenta, etc.), may be 
chosen instead of gentian- violet. It is immaterial 



REAGENTS. MICRO-CHEMISTRY. 83 

whether the dye is added to the aniline- water in sub- 
stance, or in a concentrated alcoholic solution. From 
ten to twenty drops, or more, of this solution, which 
caoi be kept on hand for weeks (although aniline- 
water itself decomposes pretty quickly when ex- 
posed to the light), are filtered off into a watch- 
glass ; the section to be examined, or the cover- 
glass, is placed in the dish for about twenty-four 
hours. The deeply-stained section, or cover-glass, 
is transferred to a dish of distilled water, in which 
it is washed ; then it is again placed in a watch- 
glass containing a solution of three parts of nitric 
acid and one hundred parts of alcohol. In a very 
short time — from three to five minutes at the ut- 
most — the decolorization has reached the proper de- 
gree, the specimen is removed to pure alcohol, and is 
then examined in oil of cloves ; the cover-glass prep- 
arations may also be washed and examined at once 
in water. If a double staining is desired, Bismarck- 
brown is employed for specimens that have been 
colored blue, methyl-blue for those that are red. 
Care should be taken that the nuclei be not too 
deeply stained, lest the tubercle-bacilli should be 
concealed. 

The decolorization can also be effected by means 
of a watery solution of nitric acid, but this must be 
considerably stronger than the other (about one to 
three), in order to act in the same time ; the more 
dilute alcoholic solution is to be preferred, since it 
does not attack metallic instruments. If the decolor- 
ization is not complete, a portion of the former dye 
again appears after washing off the acid ; this rem- 



84: THE USE OF THE MICROSCOPE. 

nant is then easily removed by the combined action 
of the acid and alcohol. The staining of the tuber- 
cle-bacilli, on the contrary, persists even after it has 
been exposed for more than a quarter of an hour to 
the action of the acid solution. 

We are indebted to Rindfleisch for an essential 
improvement in the method — namely, rapid staining 
in the presence of heat. The watch-glass filled with 
the staining solution, which contains the specimens, is 
placed in a thermostat which is kept at a temperature 
of 60° to 80° C, or better, it is held over the flame 
of a spirit-lamp or a small gas-jet, and heated until 
thick vapors arise ; then the staining of the tubercle- 
bacilli occurs in a few minutes. This process has 
rapidly come into general use, especially in the case 
of cover-glass preparations (sputa) ; it is less suitable 
for sections. The further manipulations, decoloriz- 
ing, or double staining, are then effected in the usual 
manner, at the temperature of the room. The bacilli 
of tuberculosis may be seen in this way with a power 
of three hundred, and often even without the use of 
immersion-lenses. The best objectives are requisite 
in every case, however, in order, in the more difficult 
cases, to see the fine bacilli which may perhaps be 
concealed. The careful observer will only give a 
positive opinion concerning the absence of bacilli 
in a given specimen (which opinion may often be 
extremely important) after he has exhausted the best 
optical aids at present known ; otherwise, gross errors 
would occur, as has already happened in the experi- 
ence of many. He who desires to undertake the ex- 
amination of tubercle-bacilli for diagnostic purposes 



REAGENTS. MICRO-CHEMISTRY. 85 

must accordingly be provided with an Abbe's illumi- 
nating apparatus, and a powerful immersion-lens — if 
possible, an oil-immersion. 

Unfortunately, the preparations are generally not 
permanent ; the color of the bacilli is apt to fade or 
to disappear entirely within a few months, often, still 
more rapidly. 

The tubercle-bacilli may be identified even with- 
out staining. Baumgarten demonstrated them in the 
inoculated tuberculosis of animals, and later in man 
also, by treating the sections with liquor potassse, 
even before he was aware of Koch's results. In fact, 
it is possible in many cases of tuberculosis to identify 
the bacilli in this simple manner ; however, we must 
necessarily prefer, in every case, Koch's staining 
process, with Ehrlich's modification. For by the aid 
of the latter alone do w^e decide whether we really 
have to do with tubercle-bacilli, since, as already 
mentioned, the other varieties of bacilli, with the sin- 
gle exception of those of leprosy, do not manifest this 
special behavior toward the staining process. Be- 
sides, it is an undoubted fact that in many cases in 
which the bacilli are few in number, their discovery 
is rendered much easier, or possible, by their intense 
staining. In the tubercles of fungous joint-disease, 
for example, we shall seldom succeed in seeing the 
individual bacilli by the use of the potash-method, 
whereas we almost always succeed with the staining. 
Moreover, we can never dispense with the staining- 
process in examinations undertaken for diagnostic 
purposes (sputa, pus, etc.). The colored tubercle- 
bacilli frequently show interruptions, in the form of 



86 THE USE OF THE MICROSCOPE. 

clear, unstained portions of round or oval shape, 
which, are regarded as spores. 

16. The Noble Metals, (a) Silver.— The "sil- 
ver method," introduced by von Recklinghausen, is 
of great value in normal histology ; the important 
discovery that the walls of lymph- and blood-vessels, 
formerly regarded as homogeneous, are really com- 
posed of endothelial cells, was rendered possible 
through the use of the silver process, and it is still 
almost indispensable for the demonstration of this 
fact. It is but seldom used for our purposes ; the 
most simple case is that in which it is a question of 
demonstrating an endothelial layer upon a given sur- 
face, but it is more difficult if pathological changes 
of the walls of blood- or lymph-capillaries are to be 
studied. 

The difficulties of the method consist in the fact 
that the silver salt which is employed — usually the 
nitrate — forms, with the albuminous fluids of the 
body, granular and thready coagula, which are of 
very irregular shape, and may readily form deceptive 
images ; in order to avoid this we endeavor to con- 
fine the silver precipitate entirely to the borders of 
the endothelial cells. The silver process can there- 
fore be used only with normal surfaces ; the agent 
invades to a very slight extent the interior of the 
tissues. It is better to use quite dilute solutions, 
as one to five hundred; the surface is, if neces- 
sary, washed with distilled water or with a dilute 
solution of nitrate of sodium (two per cent.), then 
the solution of silver is poured upon it, and after 
about a minute it is again washed with distilled 



REAGENTS. MICRO-CHEMISTRY. 87 

water. After a short interval (most rapidly under 
the influence of sunlight) deep black lines appear at 
the borders of the endothelial cells ; the nuclei are not 
usually colored, but they can afterward be stained 
with hematoxylin. The precipitate of silver is readi- 
ly soluble in dilute ammonia. In order to define the 
borders of the endothelial cells in the capillaries the 
silver salt is injected into an artery ; by injecting the 
solution of silver into the bronchial-tree, the borders 
of the alveolar epithelia of the lungs are stained. If 
about five per cent, of gelatin is added to the solu- 
tion while heating, we obtain a " silver cement," also 
very useful for injections, which stains the borders of 
cells lining injected cavities a brown color. If a cor- 
nea is placed for a short time in a silver-solution, or 
if it is even rubbed with the solid stick, there occurs 
a dark brownish staining of the basement-substance, 
within which the corneal corpuscles appear as bright 
radiating figures, or spaces. 

It should always be observed that the silver pro- 
cess is only applicable to fresh preparations, in which 
cadaveric decomposition has not yet begun. 

(b) Gold. — The chloride-of-gold method, which 
was introduced by Cohnheim, offers similar diffi- 
culties ; this reagent, too, penetrates only to a slight 
extent into the interior, and has besides this disad- 
vantage, that its action is not quite constant. The con- 
ditions under which the reduction of the salt, and at 
the same time the staining, take place are not yet ex- 
actly known to us. However, successful gold prep- 
arations are very valuable on account of their ex- 
tremely exact delineation, so that we can not dis- 



88 THE USE OF THE MICROSCOPE. 

pense with, this process in experiments on the cornea, 
keratitis, reproduction, etc. 

The advantages of the gold method consist in 
this : 1. The protoplasm of cells, especially in the 
cornea, is very darkly stained, and is thus sharply 
distinguished from the perfectly clear basement-sub- 
stance. 2. The axis-cylinders of the nerve-fibers are 
stained separately. Different solutions of chloride of 
gold are used, from one to one hundred to one to 
one thousand ; the cornea, or other lamellae that are 
to be stained, are left in the solution from ten min- 
utes to an hour ; this reagent also affects only the 
superficial portions of the specimen. They take a 
straw-yellow stain in this, and are then placed for a 
somewhat longer time (about twenty -four hours) in 
a dilute acid, as acetic, formic, tartaric, or citric ; 
Ranvier has recently employed lemon-juice. Then 
the reduction has either occurred, already or it takes 
place completely in the course of several days, during 
which time the specimen is preserved in alcohol or 
glycerin. The color is dark violet. The preparation 
acquires a firm consistence through the action of the 
gold salt, so that it may be cut into the thinnest sec- 
tions ; or it may be still further hardened in alcohol. 
Chloride of gold may also be used for the staining of 
sections of the nervous system, which have been pre- 
pared after the ordinary treatment with Miiller's 
fluid (Leber) ; the sections are immersed for about 
an hour in a one-half-per-cent. solution, and are then 
placed in distilled water, when they show in the 
course of one or two days a deep violet staining of 
those parts which contain the normal nerve-medulla. 



REAGEXTS. MICRO-CHEMISTRY. 89 

The method is, therefore, very useful for identifying 
degenerations and atrophies in the peripheral nerves, 
and in the white substance of the central organs. 

In order to stain the nerves with gold in alcoholic 
preparations, Frisch recommends the following meth- 
od : The sections are washed in water, are placed for 
twenty-four hours in a solution of sodium chloride 
(six-tenths of one per cent.), then for ten minutes in 
formic acid (ten per cent.) ; they are next thoroughly 
washed for from one-half to three hours in a one-per- 
cent, solution of sodium chloride and gold, while pro- 
tected from the influence of light. Then they are 
again washed and immersed in ten-per-cent. formic 
acid for twenty-four hours. 

(<?) Osmic Acid. — This reagent, which was first 
used by Max Schulze, has been employed in many 
different ways. It serves (1) for fixing and harden- 
ing delicate tissue-elements in nearly their natural 
form ; (2) for bringing out or staining fats, including 
the nerve-medulla. 

The osmic-acid solution also penetrates only the 
superficial layers of preparations. A solution having 
a strength of from one-tenth to one per cent, is used. 
It should be remembered that the fumes of osmium 
are very irritating to the conjunctiva and mucous 
membrane of the nose. The solution of osmic acid 
should be kept in brown bottles, as well as that of 
the chloride of gold and nitrate of silver. 

In small bits of fresh tissue, which have been im- 
mersed for a short time (about an hour) in dilute 
osmium solutions, and have then been mounted in 
glycerin, the cellular and fibrous elements are often 



90 THE USE OF THE MICROSCOPE. 

isolated very well, since they have received through 
the action of the reagent a certain resistance as well 
as a brown color. This method is to be especially 
recommended in the case of the nervous system ; the 
nerve-medulla is stained dark blue or black. The 
red blood-corpuscles are also colored brown by osmi- 
um, and then become quite resistant toward most in- 
fluences ; even the fumes of osmium, which are given 
off at the temperature of the room, act in this way. 
The latter reaction is effected by holding the speci- 
men, which is on the under surface of the slide, 
over the neck of a bottle filled with osniic acid. If 
the acid acts more vigorously and for a longer time, 
small pieces of tissues, as nerves, etc., become hard- 
ened. The various fats, as well as the medulla of 
nerves, are stained a deep-blue color in a few min- 
utes by the action of the acid, through the reduction 
of the metal, as is generally stated ; a special combi- 
nation probably occurs here. This striking dye 
is of great value to us, since it gives the best staining- 
reaction for fats. The reaction takes place very well, 
even in sections that have been made from alcoholic 
specimens ; that part of the fat which is not dissolved 
by alcohol is stained dark brown within a quarter of 
an hour. The osmium process can be highly recom- 
mended for the preparation of permanent specimens 
of fatty degeneration of the kidneys, liver, heart, 
granulation-tissue, and tumors, for purposes of dem- 
onstration. The sections should be mounted in gly- 
cerin. 

17. Ammonium Sulphide. (Siderosis.) — An aque- 
ous solution of sulphide of ammonium has been used 






REAGENTS, MICRO-CHEMISTRY, 91 

extensively by Quincke in pathological and histologi- 
cal examinations as a test for iron ; * the iron, con- 
tained in the interior of cells in the form of an 
albuminate, is precipitated by the ammonium sul- 
phide in the form of dark-green granules (sulphate 
of iron). The ferruginous particles are often recog- 
nized by their yellowish-brown color before the ac- 
tion of the reagent occurs, so that this is not indis- 
pensable, and, on the other hand, all yellow pigment- 
granules do not show the dark green staining with 
the reagent. The substance formerly employed as a 
micro-chemical test for iron, ferrocyanide of potas- 
sium mixed with hydrochloric acid, is less favorable 
because it coagulates albuminous bodies, and, besides, 
gives colors that are readily diffusible; the Berlin 
blue which is formed is not quite insoluble in the 
acid albuminous fluid. The dark green staining of 
the ferruginous granules with ammonium sulphide 
appears in a few minutes in sections of alcoholic 
specimens, and lasts for weeks. 

Normal red blood-corpuscles do not give the re- 
action, hence the inference is that iron is not pre- 
cipitated from all of its combinations by means of 
sulphide of ammonium. On the contrary, Quincke 
states that, even in the normal liver, and especially 
in the spleen and medulla of bones, " siderosis " is 
present — that is, ferruginous particles, which must 
have been derived from disintegrated red blood-cor- 
puscles, can be identified by the aid of the sulphide. 
The destruction of red blood - corpuscles increases 

* Quincke, "Uber Siderosis," "Deutsch. Arch, fur klin. Med.,"Bd. 
25. 



92 TEE USE OF TEE MICROSCOPE. 

very greatly as a result of transfusion (in so-called 
artificial plethora), so tliat the physiological sidero- 
sis becomes considerably extended. The particles of 
iron in the liver are contained within the capillaries 
in the interior of white blood - corpuscles ; in the 
spleen and in the osseous medulla they lie within 
the cells of the pulp. A very decided siderosis also 
occurs under similar conditions in man — that is, in 
cases in which a marked destruction of the red blood- 
corpuscles occurs, especially in pernicious anaemia. 
Iron is then demonstrable in the cells, capillaries, and 
also in the perivascular connective-tissue of the liver, 
in the gland-cells of the pancreas, in the epithelium 
of isolated looped tubules of the kidney, and also 
in the spleen and osseous medulla. 



IV. 
OTHER METHODS OF PREPARATION. 

Pkesekvatiox of Specimens. — Most of the meth- 
ods of preparing specimens have already been men- 
tioned in the former section ; the processes of hard- 
ening in alcohol and chromates, and of decalcification 
with mineral acids, have been discussed under these 
reagents. It remains to present a few of the special 
methods. 

1. Boiling. — It was formerly the custom to boil 
anatomical specimens occasionally, in order to prepare 
them for histological examination; but the method 
was first employed in a rational way by Posner * 
(from a suggestion made by the late Perls), especial- 
ly with the view of precipitating the dissolved albu- 
min rapidly and surely in loco, and thereby rendering 
it visible. 

The pieces of organs, about the size of a hazel-nut 
or walnut, are thrown into boiling water, from which 
they are removed in a few minutes, and washed in 
cold water. They have then, as a rule, a somewhat 
tough, elastic consistence, and can be at once cut with 
the razor, or they may be perfectly hardened in alco- 
hol. The coagulated albumin appears in such prepa- 

* Posner, " Virch. Arch.," Bd. 79, S. 311. 



91 THE USE OF THE MICROSCOPE. 

rations, as a coarsely granular mass ; most of the cell 
outlines also have become sharp and distinct. 

The method possesses peculiar advantages for the 
examination of kidneys in cases of albuminuria and 
of oedematous lungs. If such organs have been har- 
dened simply in alcohol, it is also possible to identify 
the granular coagulated albumin in the interior of the 
Malpighian capsules, or alveoli, especially in the su- 
perficial portions of the specimen, which were most 
directly exposed to the action of the alcohol. How- 
ever, this is effected much more perfectly by means 
of the boiling process, by which a rapid and complete 
coagulation is attained in a very short time. Aside 
from this action, most structures are only slightly 
changed by a brief exposure to a boiling tempera- 
ture. 

The boiled specimens can also be cut with the 
freezing microtome. 

2. The Dkytstg of preparations, in order to ren- 
der them suitable for cutting, was once much prac- 
ticed. Since the general introduction of the art of 
hardening in alcohol (in the case of porous or very 
soft objects, after previous saturation with mucilage), 
drying has been almost entirely given up. Prepara- 
tions shrink a great deal during the latter process ; 
but the sections swell up again in water, although 
very irregularly. 

For the purpose of macerating or isolating certain 
tissue elements, we employ 

3. Artificial Digestion, which is occasionally 
used for pathological examinations, but more espe- 
cially in normal histology. We use pepsin or tryp- 



OTHER METHODS OF PREPARATION. 95 

sin — that is, artificial gastric juice, or extract of pan- 
creas. 

Artificial gastric juice is best prepared from the 
mucous membrane of the fundus of the pig's stomach. 
The pieces of mucous membrane are chopped fine, and 
are soaked for about an hour in very dilute hydro- 
chloric acid (one to one thousand), which is placed 
in an incubator kept at the temperature of the body ; 
the mixture is then filtered. Commercial pepsin may 
also be used ; but, in any case, the digestive power 
of the fluid must be tested on bits of fibrin, or a piece 
of loose, coagulated albumin. These should be dis- 
solved in a short time. 

The extract of pancreas is prepared as follows : * 
The pancreas of a freshly killed bullock is chopped 
in pieces and completely extracted with alcohol and 
ether in an extracting-apparatus ; the white, friable 
mass that remains after the evaporation of the ether 
is digested for about four hours, at a temperature of 
40° C, with five to ten times its weight of salicylic 
acid (one-tenth of one per cent.), or with distilled 
water, and is then strained and filtered. 

The artificial gastric juice digests in a short time 
(at the temperature of the body) connective tissue, 
muscle, most cellular elements, etc., while elastic tis- 
sue and nerve-fibers resist it. Extract of pancreas, 
on the contrary, or the trypsin which it contains, 
dissolves in an acid fluid elastic fibers, as well as the 
fine fibers of the neuroglia, while the connective-tis- 
sue fibrillaB remain intact. 

* Kuelme, " Yerhandl. des med.-naturf. Yereins zu Heildelberg," I, 
1877. 



96 THE USE OF TEE MICROSCOPE. 

Digestion may be carried on either in the incu- 
bator, or (by means of the warm stage) in the micro- 
scopic specimen itself, under the eyes and the con- 
stant control of the observer. It has been recently 
discovered, by the aid of this method, that the gray 
fibrillary substance, which is found in such quanti- 
ties in the posterior columns of the spinal cord in 
tabes, corresponds perfectly in its chemical composi- 
tion with the fibers of the neuroglia ; it is also rap- 
idly dissolved, while the fibers of the pia and their 
processes remain intact (Waldstein and Weber}.* 
The so-called neurokeratin of Kuehne resists tryp- 
sin perfectly; it remains in the interior of nerve- 
fibers in the form of a delicate net-work, when 
these are extracted with hot ether and chloroform, 
and then with trypsin. The horny net-work was 
regarded by Kuehne, and by many of his followers, 
as a preformed structure — " the horny basis of nerve- 
fibers." Hesse and others oppose this view, more re- 
cently also Waldstein and Weber (pupils of Ran- 
vier). 

These authors assert that neurokeratin is mingled 
in quite a diffuse manner with the medulla, and that 
it only assumes its peculiar net-like structure during 
the process of extraction ; the form of the net- work 
can be varied at will according to the variation of 
the process. By similar treatment the same " horny 
network " can be produced in the irregular drops of 
the escaped medulla (myelin) as in the interior of 
nerve-fibers. Neurokeratin disappears in degenerat- 

* " Arch, de physiolog. norm, et pathol.," II. Reihe, Bd. 10, 1882, 
S.l. 



OTHER METHODS OF PREPARATION. 97 

ed nerves, as well as in gray degeneration of the 
white substance of the central nervous system. 

4. Imbedding. — Most of our hardened specimens 
are cut without imbedding. In speaking of the mi- 
crotome, allusion was made to the little devices of 
gluing the preparations upon cork, and of securing 
them between pieces of hardened liver ; we are thus 
able in most cases to fix the specimens sufficiently 
without further imbedding, so as to obtain perfect, 
even sections. If we have an irregular surface to 
cut, and it is desirable that the sections should in- 
clude the surface itself, as in the examination of a 
menstrual mucous membrane, a thin layer of muci- 
lage is applied to the surface in question, and upon 
this is placed a slice of hardened liver. The gum 
soon hardens in alcohol, so that a firm union be- 
tween the liver and surface of the preparation oc- 
curs. If the inequalities of the surface are con- 
siderable, the mucilage is less useful, since in thicker 
layers the gum acquires a stony hardness, so as to 
injure the knife ; glycerin-cement is used in this case, 
after the example of Klebs. This is prepared in 
the following manner : Ten grammes of the finest 
well-washed gelatin are allowed to soak in distilled 
water, the residue of water is poured off, and the 
swollen glue is dissolved by gentle heat ; to this 
add ten grammes of glycerin, and also a few drops 
of phenol to prevent the formation of mold. A 
small part of this mass, which becomes solid at the 
temperature of the room, may readily be dissolved 
by warming at each time of using; the irregular 
surface of the preparation is covered with the fluid, 



98 THE USE OF TEE MICROSCOPE. 

and assumes a proper consistence for cutting wlien 
placed in alcohol. 

In other cases, however, we are compelled to cut 
a specimen in toto — that is, to include all the sur- 
faces of the same in the section — a task which very 
often devolves upon zoologists and embryologists, 
but upon us only in exceptional cases. The speci- 
men must then be surrounded with a solid mass; 
glycerin-cement can also be used for this purpose, but 
it is better to employ a substance which does not 
shrink when it hardens, and even when it is treated 
with alcohol. We use, for example, a mixture of 
equal parts of w^ax and oil, or two parts of sperma- 
ceti and one part of oil of bergamot, or the follow- 
ing : 

Paraffin 5 parts. 

Spermaceti 2 " 

Suet 1 part. 

The mixture liquifies when heated gently, is poured 
around the alcoholic preparation (which has been 
placed in a tin box, properly adapted to the micro- 
tome-clamp), and, after cooling and an additional stay 
in alcohol, it again solidifies. If we wish the fat to 
penetrate the specimen, the latter must of course be 
completely dehydrated ; it is usually immersed in an 
ethereal oil, as oil of bergamot, before it is saturated 
with the fat. In this way the specimens acquire an 
exceedingly even consistence; the fat is afterward 
removed from the sections. As is apparent, the pro- 
cess is somewhat lengthy ; it will be very seldom 
necessary to employ it for our purposes. 

Another favorite imbedding material is Caberla's. 



OTHER METHODS OF PREPARATION. 99 

This consists of fifteen parts of white of egg and 
one part of a ten-per-cent. solution of carbonate of 
sodium ; the accompanying yolks are added, and the 
mixture is shaken. The object is placed in a paper 
box, which stands in a dish filled with alcohol having 
a strength of eighty per cent., and the dish is then 
heated on the water-bath up to about 75° C. ; after 
warming for half an hour, coagulation has occurred 
to a sufficient extent, and the specimen is hardened 
in alcohol. 

These imbedding substances are opaque, so that 
marks must be placed on their surfaces in order to 
show the position of the preparation. 

The most valuable imbedding material is cer- 
tainly celloidin, for the introduction of which into 
microscopical technology we are indebted to Schief er- 
decker ("Arch, fur Anat.," 1882). Celloidin is a 
substance resembling collodion, and is sold in solid 
masses. It dissolves slowly in a solution of alcohol 
and ether, forming a sirupy liquid. If the alcoholic 
preparation is now immersed in this, it becomes 
thoroughly saturated with the celloidin solution in 
the course of several hours (about twenty-four). The 
specimen thus filled with celloidin is then placed in 
seventy or eighty per cent, alcohol, in which the cel- 
loidin again solidifies, so that the specimen has a very 
uniform consistence for cutting. It is next trans- 
ferred to the microtome, enveloped as it is in a near- 
ly transparent mantle of celloidin, so that every sec- 
tion is surrounded by a like covering, and can be 
stained, examined, and mounted together with this. 
Oil of cloves is not used as a clearing agent in this 



100 THE USE OF THE MICROSCOPE. 

case, since it dissolves celloidin, but oil of origanum 
or cedar. 

The celloidin method has been approved in many 
instances ; it is especially valuable for eyes, the 
spinal cord, etc. 

5. Process of Injection. — This is employed far 
less frequently in pathological examinations than in 
normal histology. 

It is highly important that we should study the 
natural injection of the blood-vessels with blood, as 
well as the lymph-vessels with lymph, and we seldom 
resort to an artificial filling of the lumina. Hence, 
only a brief sketch of the rather complicated process 
of injection is presented, while those who are inter- 
ested in the subject are referred to the well-known 
text-books of Ranvier and Frey. 

(a) Injecting Material. — We use as an injecting 
material a transparent but deeply colored fluid, such 
as solidifies within the vessels, generally a glue ; in 
using the latter, the injecting fluid and the organ to 
be injected must be raised to rather a high tempera- 
ture (40° or 50° C). For this purpose large tin 
dishes are used, which are filled with water and 
heated from beneath. Glue injections are therefore 
rather troublesome, but they are preferable to watery 
solutions, on account of the stability of the material. 

Soluble Berlin-blue, which can be obtained from 
druggists, serves for coloring the mass ; sometimes 
this only dissolves after the addition of a little oxalic 
acid. A solution of this material in from ten to 
twenty parts of water may be used directly for in- 
jecting, or five parts of alcohol and five of glycerin 



OTHER METHODS OF PREPARATION. 101 

may be added. Or, on the other hand, the hot, 
watery solution of Berlin-blue may be poured grad- 
ually into the same quantity of hot, concentrated 
solution of glue, while stirring constantly ; the latter 
is prepared by allowing fine, well- washed sheets of 
glycerin to soak for one or two hours, at the tempera- 
ture of the room, in about double the quantity of 
distilled water ; the swollen glue is then liquefied by 
gentle heating upon the water-bath. 

Since the " soluble Berlin-blue" of the druggists 
is not always reliable, we shall repeat here the exact 
direction of Thiersch for preparing the substance in- 
dependently.* 

Thiersch's Berlin-blue, prepared with Oxalic Acid. 
— Make a cold, saturated solution of sulphate of iron 
(A), a similar one of ferrocyanide of potassium (B), 
and, thirdly, a saturated solution of oxalic acid (C). 
Finally, a warm, concentrated solution (two to one) 
of rather fine glue is needed. About fifteen grammes 
of the solution of glue are mixed with six cubic 
centimetres of solution A in a porcelain dish. In 
a second larger dish thirty grammes of the solution 
of glue are mixed with twelve cubic centimetres 
of solution B, and to this twelve cubic centimetres 
of the oxalic -acid solution C are subsequently 
added. When the mass in both dishes has cooled to 
about 25° or 32° C, the contents of the first dish are 
added, drop by drop, to the mixture in the second 
while agitating constantly. After complete precipi- 
tation the resulting dark-blue substance is heated for 
some time at a temperature of 70° to 100° C, while 

* From Frey, " Das Microscop," 1881. 



102 THE USE OF THE MICROSCOPE. 

stirring, and is finally filtered through flannel. The 
injections of Berlin-blue are beautifully colored, but, 
in the course of time the color gradually disappears 
as a result of reduction; the blue dye is again re- 
stored through the action of an ozone-carrier, as oil of 
turpentine. 

Injections of carmine, on the contrary, are quite 
permanent ; but here we encounter the difficulty that, 
in using an alkaline solution of ammonia-carmine, the 
red color at once transudes ; the solution must, there- 
fore, be neutralized. The neutralization must be 
effected with extreme care, since the material other- 
wise becomes opaque and perfectly useless, by reason 
of the coarse precipitate of carmine. 

Cold Fluid-injection of Carmine. — One gramme 
of carmine is dissolved in a little water with one 
gramme of ammonia, and to this mixture twenty 
cubic centimetres of glycerin are added. To this so- 
lution is added carefully a mixture consisting of 
twenty cubic centimetres of glycerin and one cubic 
centimetre of hydrochloric acid, while stirring vio- 
lently ; the whole is then diluted with forty grammes 
of water (Kollmann). 

Fretfs Carmine-cement. — Take a solution of am- 
monia and one of acetic acid, the number of drops 
of which necessary for ready neutralization have been 
previously determined. From two to two and a half 
grammes of the finest carmine are dissolved in a dish 
by stirring with a fixed number of drops of the am- 
monia solution (which can be increased or diminished 
at pleasure), and with about fifteen cubic centimetres 
of distilled water, and the solution is then filtered ; 



OTHER METHODS OF PREPARATION. 103 

several hours are required for this purpose and a loss 
of ammonia results through evaporation. The alka- 
line ammonia-carmine solution is added to a filtered, 
moderately warmed, concentrated solution of fine 
glue, while agitating ; the mixture is heated for a 
while upon the water-bath, the number of drops of 
acetic acid necessary to neutralize the original solu- 
tion of ammonia are added slowly, and the whole is 
stirred. Thus the carmine is precipitated in the acid 
solution of glue. 

In injecting organs that have a pretty strong alka- 
line reaction, a small quantity of acetic acid may still 
be added to the material. During the injection the 
temperature should not exceed 45° C. 

Other substances also have recently been em- 
ployed for injecting the finest lymph-spaces, such as 
oily fluids which are colored with alkanet, generally 
oil of turpentine, or even chloroform in which a dark 
resinous body, as asphalt, is dissolved. 

(V) Injecting Apparatus. — An injecting-syringe 
is often employed, which must work well and be 
very carefully cleansed after using; the syringe is 
connected with the canula, either directly or by 
means of a rubber tube. The syringe and canulae 
are made of metal or glass ; the latter we can easily 
prepare for ourselves of any desired shape. The 
piston of the syringe must not fit too tightly, and 
must move quite smoothly, without jerking. 

Injection under constant pressure is an under- 
taking rather more complicated, but is one to be 
highly recommended. If the large machine of Her- 
ing is not at hand for this purpose, the necessary ap- 



104 THE USE OF THE MICROSCOPE. 

paratus can be easily arranged by means of some 
wash-bottles and rubber tubes. The bottles are 
closed by rubber stoppers, perforated in two places, 
each of which is armed with two glass tubes in the 
ordinary manner, the short, thick one ending just 
below the stopper, while the other long one reaches 
down to the bottom of the bottle. One bottle, A, is 
nearly filled with the injecting-fiuid ; the long glass 
tube which dips into the fluid is connected at its other 
end with the injecting-canula. The other short tube 
is connected with the second bottle, B, which serves 
as an air-chamber, and at first contains nothing but air. 
This second bottle is again attached, by means of a 
long rubber tube and the glass tube that extends to 
the bottom, with a pressure- vessel, (7, which can be ele- 
vated at will (by placing blocks under it) and is filled 
with mercury. If, now, the mercury is allowed to flow 
from the pressure-vessel, C, into the bottle, B, which 
represents the air-chamber, the air-space ifi^fi^is sub- 
jected to a pressure corresponding to the difference 
in level of the mercury in the two vessels; this 
pressure is at once transmitted to the injecting-fluid 
contained within the bottle A, and it is forced into 
the lumen of the vessel under the same pressure. 
Care is to be taken that the difference in level of 
the two portions of mercury remains the same. If 
the pressure-bottle, (7, is elevated according as the 
fluid gradually flows out, the injecting-pressure re- 
mains constant. If there is a bulb-apparatus at hand, 
this may be substituted for the pressure-bottle. The 
pressure-vessel can be filled with water instead of 
mercury ; in order to attain a greater pressure, the 



OTHER METHODS OF PREPARATION. 105 

latter is placed upon a cupboard, etc. Of course, 
all tlie connections must fit accurately and in an 
air-tight manner. The canula, which is tied in the 
vessel, is filled with the injecting-material, or with 
distilled water, in order to avoid impurities, and is 
attached to the syringe, or other apparatus, in such 
a way that no air-bubble shall enter ; the injection 
can then be begun. Moreover, the stream of inject- 
ing-fluid must never be interrupted by air-bubbles ; 
they can certainly be avoided by carrying out the 
preparations carefully. 

If the injection is taking place, the fluid soon 
escapes from the lateral or adjacent branches, es- 
pecially if the organs, or portions of organs, have 
been removed at an autopsy before the injection was 
contemplated ; the vessels must then be tied. It is 
better to leave the veins open at first, so that the 
blood can escape ; these may also be ligated before 
the process is finished. By choosing proper arterial 
branches valuable injections can be made even in 
kidneys, lungs, livers, etc., which have already been 
cut through ; this will be limited, however, to por- 
tions of the organ. * The injection is discontinued 
when the staining of the organ is sufficiently in- 
tense ; the increase in the consistence of the parts 
also furnishes an indication as to when it is neces- 
sary to stop. After being injected, the specimens 
are at once transferred to alcohol. 

During the injection of organs that have been 

* Rindfleisch uses for this purpose thin elastic catheters, which are 
inserted deeply into the interior of organs, like injecting-canulae ; small 
openings are made at their extremities. 



106 THE USE OF THE MICROSCOPE. 

subject to pathological changes, we must frequently 
contend with extravasations ; these can be disregard- 
ed, at least in many cases, by avoiding excessive press- 
ure. In injecting lymph-vessels we frequently use 
delicate needle-canulao (the so-called " puncture-injec- 
tions "), which are carefully thrust into the organ at 
the desired spot ; an " extravasation " necessarily oc- 
curs in the neighborhood of the injection, but the 
lymph-vessels are often filled most beautifully, and 
that, too, in a very simple and rapid way. 

The methods of performing injections for physi- 
ological purposes have been greatly improved and ex- 
tended during the past few years by Cohnheim, Hei- 
denhain, Arnold, Thoma, and others. These have at- 
tained much importance in connection with many 
questions in pathology, especially in the case of the 
kidneys. However, they are only important in ex- 
perimental investigations; we can not enter into a 
discussion of them here. 

6. Preservation of Specimens. — In order to pre- 
serve for a few days fresh specimens that lie in salt- 
solution, it is only necessaiy to place them in a moist 
space. This is very easily arranged in the following 
manner : A large, flat dish is filled to the height of 
several millimetres with water; in the dish stands 
upon three feet (formed of corks attached to it by 
means of sealing-wax) a small glass plate, which sup- 
ports the specimens ; a bell-glass, lined on its interior 
with damp blotting-paper, is inverted over this so 
that in this way the space in which the specimens are 
preserved is kept closed and sufficiently moist. In- 
stead of the glass plate a wooden or metal etagere 



OTHER METHODS OF PREPARATION. 107 

may be used, in which the preparations are placed in 
several layers one above the other without touching ; 
or a number of these plates or tables may be built up, 
one over the other, an antiseptic being added to the 
surrounding water. Fresh specimens do not preserve 
their perfect delicacy very long ; it is of no use to 
seal them up in salt-solution, since the elements are 
soon destroyed, as a rule ; and besides, the water gen- 
erally evaporates in spite of care in cementing. Many 
objects can be preserved in acetate of potash, a satu- 
rated solution of which is known to be stable in the 
air ; but in this, too, much of the original delicacy of 
the outlines is lost. 

The most important question is the preservation 
of sections made from alcoholic specimens. If the 
sections are mounted in glycerin, they are already 
sufficiently preserved, and it is only necessary to 
fasten the cover-glass so that dust, or any other im- 
purity, which collects upon it, can be wiped away. 
For this purpose the superfluous glycerin is pressed 
out by placing a bullet upon the cover-glass, and 
is absorbed by blotting-paper ; then the slide is care- 
fully cleansed around the cover-glass — that is, every 
adherent trace of glycerin is removed, which is best 
done by means of fine linen moistened with alcohol. 
The edge of the cover-glass is then surrounded 
with some hardening cement, and is thus fixed to 
the slide. Canada balsam in chloroform, or Bruns- 
wick-black, mask- varnish (Maskenlack), etc., is used 
as a cement. Following Ranvier's suggestion, I 
have long employed a thick solution of good red 
sealing-wax in alcohol. For glycerin we may sub- 



108 THE USE OF THE MICROSCOPE. 

stitute glycerin-cement, which liquefies on warming 
slightly and hardens on cooling. Or gum-arabic is 
added to the glycerin, so that the layer at the edge 
gradually becomes solid. Farrant's mixture, consist- 
ing of equal parts of glycerin, gum-arabic and a 
saturated solution of arsenious acid, is worthy of 
recommendation. For most cases simple mucilage is 
sufficient ; the specimens are inclosed in the sirupy 
liquid, the latter dries up at the edge, and without 
any further care the sections are permanently fixed 
and preserved. 

Preparations that have been cleared in oil of 
cloves are mounted in Canada balsam, as was pre- 
viously stated (page 40) ; here, too, further cement- 
ing is unnecessary. 



V. 

THE OBSERVATION OF LIVING TISSUES. 

The Cikculation. Inflammation. — The obser- 
vation of pathological processes in living tissue by 
means of the microscope is practicable only to a very 
limited extent in man; the instrument devised by 
Hiiter for observing the circulation in the mucous 
membrane of the cheek, and in similar regions, fur- 
nishes very defective images. In warm-blooded ani- 
mals, also, the difficulties are considerable ; however, 
these have been overcome, for Strieker and Thoma 
have constructed complicated instruments which al- 
low of the observation in mammalians of the circu- 
lation, of its pathological disturbances, and of in- 
flammation. Systematic artificial respiration is ne- 
cessary, otherwise the curarized animals would soon 
die ; beside this, the exposed transparent part which 
is being observed microscopically (the mesentery is 
the best) must always be kept at the temperature of 
the animal's body. All these precautions, however, 
have not hitherto led to any very important results ; 
our knowledge has not been essentially advanced 
through careful investigations made upon warm- 
blooded animals. The well-known, striking processes 
were firs # t demonstrated in the cold-blooded animal 



110 THE USE OF THE MICROSCOPE. 

(the frog), principally by Colmlieim. On account of 
the great importance of the observations in question 
the very simple methods necessary for making them 
will be briefly described. In order to observe the 
circulation and its disturbances in frogs it is well to 
paralyze the voluntary movements of the animals by 
means of curara. If a particle of curara one half to 
one millimetre in diameter is placed under the skin 
of a large frog, the animal becomes motionless in the 
course of half an hour, while the vegetative functions 
continue. The frog's small need of oxygen can be 
supplied for days through the cutaneous respiration 
alone. Three regions in particular can then be util- 
ized for the study of the circulation. 

1. The Web. — The web possesses the advantage 
that it is not necessary to inflict an injury in order to 
observe the vital processes, since it is enough to sim- 
ply separate two toes and to fix them apart. This 
part is therefore very valuable for many observa- 
tions ; but it is inferior in transparency to other ob- 
jects soon to be mentioned. Although animals are 
selected which are as poor in pigment as possible, the 
pigment-cells, as well as the sharp outlines of the 
numerous layers of pavement epithelial cells, are 
troublesome. Besides, real inflammatory processes 
extend merely to a slight distance in the dense tis- 
sue; only disturbances of the circulation, vascular 
dilatation and contraction, or even necrosis, are 
caused by the action of various irritants, but inflam- 
matory swellings are not generally absent. 

2. The Tongue. — The tongue is drawn out of the 
mouth, stretched over a ring of cork, and fastened 



THE OBSERVATION OF LIVING TISSUES. HI 

to it by fine insect-pins, or hedgehog-spines which 
are then cut off short. Without further preparation 
it is generally too opaque for examination with strong 
lenses; a small piece is removed with fine scissors 
from the upper (originally the lower) surface. By 
avoiding the vessels that are visible, the escape of 
blood is prevented as much as possible ; the blood is 
then washed away with salt-solution. If the field is 
clear, the cover-glass is adjusted ; drying is prevented 
by dropping salt-solution upon the part, and the rest 
of the frog's body is wrapped in moist blotting-paper. 
The frog and the cork ring that supports the tongue 
are placed upon a glass plate, which is moved di- 
rectly under the microscope. The observation can 
then be begun and continued for hours, and even for 
days. The tongue must not be stretched too much, 
lest an obstruction to the flow of blood result. 

An object thus prepared is also perfectly suitable 
for strong lenses; the escape of the white aod red 
blood-corpuscles can be demonstrated very well here, 
an artificial wound serving as an irritant to provoke 
inflammation. 

3. Mesentery. — Use large male frogs (recognized 
by the glands on their thumbs), so as not to be em- 
barrassed by the oviducts and ovaries. Incise the 
skin in the axillary line over the lower half of the 
trunk ; the resulting haemorrhage soon ceases, the 
incision is then carried through the muscles, and the 
abdominal cavity is opened for a distance of from ten 
to twenty millimetres. A coil of small intestine is 
carefully drawn out with blunt forceps, and is 
stretched over a cork ring and secured in the manner 



112 THE USE OF THE MICROSCOPE. 

described for the tongue. The mesentery must not 
be stretched too tightly, otherwise obstruction re- 
sults ; it is then covered with a piece of cover-glass, 
and presents a brilliant object for examination with 
the strongest objectives. The mesentery is kept 
from drying by the use of salt-solution, while the frog 
is enveloped in a damp covering. Here also the cork 
ring is not cemented to the glass plate, but it is much 
more convenient to leave it free. The more care- 
fully the specimen is prepared, and the more all ten- 
sion and other mechanical injury are avoided, so much 
the longer is it preserved, until exquisite inflamma- 
tory phenomena, arrangement of the white blood-cor- 
puscles along the walls of the vessels, emigration, etc., 
take place. In the case of the prepared tongue, as 
well as in the mesentery, we can apply any desired 
irritant, or cause any injury to the tissue, as well as 
to the vessels. 

The lungs and urinary bladder of the frog may be 
easily prepared for microscopical examination in a 
similar manner. 

4. Cornea. — The lon^-lived cornea of the fros; 
furnishes a good object for the observation of patho- 
logical processes, especially inflammation. The cor- 
nea, normal or inflamed (after cauterization, for ex- 
ample), is carefully excised and is placed upon the 
slide in the drop of aqueous humor which escapes at 
the time ; the edge is then nicked in several places, 
so that the membrane may be spread out smoothly. 
The vital appearances of the cells, the wandering as 
well as the fixed, can be observed for several hours 
in the cornea after its removal. 



VI. 

THE EXAMINATION OF FLUIDS. 

The microscopic examination of fluids is extreme- 
ly valuable for clinical and pathological purposes ; a 
glance at the microscope frequently establishes the 
positive diagnosis of a disease which was previously 
obscure or misunderstood. The process offers but 
few technical difficulties, since it is simply a question 
of transferring a small drop of the fluid to the slide 
by means of a glass rod, and covering it with a 
cover-glass. The drop must not be so large that 
the fluid flows over the edge of the cover-glass, nor 
so deep that the glass floats upon it — this is all self- 
evident. 

Our task now consists in examining the morpho- 
logical elements contained in the fluid. These in a 
great many cases may be demonstrated at once with 
the naked eye, either in the form of a diffused cloudi- 
ness, or as coarser shreds or granular precipitates. 
These latter objects are, of course, first utilized for 
microscopical examinations, being collected with a 
small spoon, or a pair of forceps, and examined with 
powers of gradually increasing strength. The sub- 
stance should always be first subjected to a careful 
microscopic examination by transmitted, as well as by 



114 THE USE OF THE MICROSCOPE. 

direct light, a rule which, self-evident as it sounds, is 
however too often disregarded by the beginner. 

If a very small number of formed elements are 
present that layer of the fluid is examined in which 
they are most numerous, generally the sediment, since 
the elements are in most cases specifically heavier 
than the fluid ; only the fats float on the surface. It 
is less advisable to filter the fluid and to collect the 
residue from the filter, because in this way impuri- 
ties can not be certainly avoided. In other cases the 
morphological elements are so abundant that an ex- 
tremely thin layer must be used, in order to pre- 
vent them from lying in several strata, one above 
the other, and thus concealing one another. It is 
necessary to dilute very thick and pultaceous fluids 
in order to render the examination possible ; serum, 
or as a rule, salt-solution, having a strength of 
three-fourths of one per cent., being used for this 
purpose. 

The Vital Properties of the Suspended Elements. 
Amoeboid Movements. — Aside from this last instance, 
we also encounter the elements in their normal men- 
struum, and can therefore be sure that they appear 
as little changed as possible, provided that all unfa- 
vorable influences are avoided. The various precau- 
tions must be especially observed if we intend to 
study the vital phenomena of the elements suspended 
in the fluid. The pressure of the cover-glass is next 
to be considered, since this may become considerable, 
not only by reason of the weight of the glass, but 
still more through the capillary attraction exerted 
in a thin layer of liquid; it must accordingly be sup- 



THE EXAMINATION OF FLUIDS. 115 

ported, if necessary, by placing beneath it bits of 
glass, such as fragments of covers. 

Furthermore, the fluid must be prevented from 
evaporating, and thus changing its concentration; 
this evil results very quickly at the edge of the 
specimen, but more slowly in the center, so much 
more slowly in fact the deeper the layer of fluid and 
the further we are removed from the edge. The 
evaporation may be reduced to a minimum by placing 
the preparation in some sort of moist cell. Take sim- 
ply a wide glass tube, about two or three centimetres 
high, as a piece of a lamp-chimney ; the interior of 
this is lined with a thick layer of moist blotting- 
paper, and the glass is placed over the specimen as it 
lies upon a broad slide ; the opening is almost entirely 
closed above by the tube of the microscope. 

It is better to examine the fluid in the form of a 
suspended drop, by using slides with cells that have 
a depth of one or two millimetres; the latter are 
easily constructed by cementing glass rings or bor- 
ders to the slides, or they may be obtained from op- 
ticians under the name of " hollow-ground slides." If 
then the edge of the cell is oiled, and the drop of 
fluid is placed in the middle of the cover-glass, upon 
its under surface, a hermetically-sealed space is 
formed, within which no further evaporation occurs. 
This is the manner in which fluids must be examined 
in order to study the motor phenomena of the con- 
tained schizomycetes. If two tubes then open into 
the cell, we can study under the microscope the influ- 
ence of gases upon the elements contained within the 
suspended drop (gas-cell). The so-called " amoeboid " 



116 THE USE OF THE MICROSCOPE. 

protoplasmic movements, as well as tlie processes of 
division in living cells may be observed in this way ; 
during this examination all currents in the fluid itself 
must be avoided, lest a whirling about of the ele- 
ments be mistaken for a change in their shapes. The 
colorless cells of the blood and lymph, pus- and mu- 
cus-corpuscles, many cells met with in exudations, 
and even tumor-cells, afford an opportunity for these 
highly interesting and engaging observations. He 
who intends to undertake such examinations must 
proceed ever with the most painstaking care, and 
must set to work critically, but above all with great 
patience. The movements are nearly always very 
slow, even when the warm slide is used ; Strieker's 
model of the latter is to be recommended. 

The Form of the Elements. — We are, however, 
mostly concerned with elements the form of which is 
perfectly constant, so that we have simply the task 
of studying these accurately. To this end it is neces- 
sary to view the body in question from all sides ; for 
it is quite clear that a circular figure, for example, 
which is observed under the microscope may repre- 
sent either a disk, a sphere, a cylinder, or a cone ; 
even an ellipsoid, an oval, or a still more irregularly 
shaped body, may, under certain circumstances, ap- 
pear in the microscopical image as a circle. We are 
aided here first of all by the micrometer-screw, since 
we obtain by using it the outline of the observed 
object at different focuses, and thus form a combined 
stereoscopic image; also by the passive movements 
which we can cause the object to make by allowing 
it to revolve around its different axes ; the simplest 



THE EXAMINATION OF FLUIDS. H7 

way to effect this is by exciting a current in the 
fluid, either by means of a bit of absorbent blotting- 
paper placed at the edge of the cover-glass, or by 
pressing upon the cover with a needle. The beginner 
will sometimes during the course of these manipula- 
tions, not only turn and roll the body in question, 
but will cause it to disappear entirely from the field 
of vision ; however, he soon acquires the necessary 
delicacy in graduating the pressure, and can then dis- 
criminate sharply concerning the forms of elements 
as seen from different aspects and thus easily deter- 
mine their stereometrical figures. 

The Examination of Tissue-fluid, etc. — In exam- 
ining fresh organs it is often of great importance to 
promptly observe the isolated elements (cells, etc.) 
of the same ; in many cases this is accomplished in 
an extremely simple manner by examining the tissue- 
fluid which has been wiped away. For this purpose 
a freshly cut surface is always exposed, and this is 
scraped with the blade of a scalpel. In proportion 
to the stability of the elements on the one hand and 
the firmness of their cohesion (or that of the cement- 
substance) on the other, we succeed, by employing 
firmer or lighter pressure, in isolating in this simple 
and very convenient way the elements of most paren- 
chymatous organs, or at least some of them. We 
must of course always bear in mind the narrow lim- 
its of the method ; but much time may be saved in 
this manner, since it is often by no means necessary 
in order to answer certain questions to examine accu- 
rate sections of the organ under consideration, that is, 
if the isolated elements are sufficiently characteristic. 



118 THE USE OF THE MICROSCOPE. 

The tissue-fluid that has been scraped off must 
usually be diluted before it is examined microscopi- 
cally ; salt-solution is used for this purpose as a rule. 
A fine glass capillary tube can be inserted into many 
soft tissues and the tissue-fluid with its suspended 
elements be drawn into it ; E. Neumann employs 
this method particularly in examining the lymphoid 
cells of marrow. The elements are thus always ob- 
tained in their natural menstruum. It is possible to 
isolate the elements of soft tissues in a very simple 
manner by lightly teasing with needles ; the bit of tis- 
sue is rapidly torn into small pieces in a drop of salt- 
solution. The fluid is thus filled with the separate cel- 
lular elements that are removed from the fragments ; 
these elements are examined as they float freely in the 
fluid, and so are all of the bits of tissue which have 
become sufficiently transparent, at least at their edges. 
In the case of fibrous tissues, as muscles and nerves, 
the elements are isolated in the direction of their ver- 
tical axes by careful teasing with needles. 

The Examination of Micro-organisms. — On ac- 
count of the great and ever-increasing importance, as 
well as the peculiar character of the subject, it is ad- 
visable to discuss separately the examination of fluids 
for micro-organisms, especially for schizomycetes. 

First of all it is evident that in these investiga- 
tions every impurity must be rigidly excluded ; care 
must be taken in obtaining the fluid to insure abso- 
lute cleanliness of the vessels, canulse, etc. Further- 
more, the objects must always be examined when they 
are perfectly fresh ; micro-organisms can develop in 
great numbers within a few hours since their germs 



THE EXAMINATION OF FLUIDS. 119 

are everywhere diffused. The latter are present on 
the sides of every vessel, no matter how clean it is, 
and on every wiping-cloth ; in smaller numbers also 
in the atmosphere, especially in inhabited rooms 
(hospital-wards, laboratories, etc.), so that it requires 
special precautions, such as the prolonged heating of 
all vessels to above 100° C, in order to collect and to 
preserve fluids without contamination with the acci- 
dental germs of minute organisms. The generatio 
equivoca of cleft-fungi, the doctrine of which was re- 
peatedly revived a few years ago, always resulted 
from the neglect of some one of the necessary pre- 
cautions. 

The fluid is always examined in a perfectly fresh 
condition, that is immediately after its removal from 
the living or dead body ; in the latter case we must 
ever bear in mind the possibility of a post-mortem 
origin. A method introduced by R. Koch is strongly 
recommended: the sample of fluid is removed and 
transferred to the slide by means of a platinum wire 
cemented to a glass rod, since the former can be very 
easily and surely cleaned immediately before and after 
using by heating it. 

The fluid is first examined directly, without the 
addition of any reagent, because in this way we are 
quite sure that any organisms which may be found 
really belong to the fluid itself. In many cases the 
organisms are recognized by their active movements. 
But it is necessary to use great care in this connec- 
tion, because small bodies suspended in liquids nearly 
always show, under certain circumstances, a very 
active movement — the Brownian molecular motion. 



120 THE USE OF THE MICROSCOPE. 

We do not as a rule f oitq a proper conception of the 
energy of these movements, which are for the most 
part due to currents caused by evaporation. In order 
to obtain an idea of them sprinkle some finely pow- 
dered carmine into a drop of water, and examine this 
with a high power ; you will be extremely surprised 
at first at the rapidity and apparent spontaneity of 
the passive movements of the carmine granules. Be- 
fore, then, an opinion is hazarded concerning the 
" spontaneous movements " of granules which you are 
inclined to call micro-organisms, it is strongly ad- 
visable to familiarize yourself perfectly with Brown- 
ian molecular motion. Even if you think that you 
have to do with vital movements, you must always 
prove this by showing that the motion ceases when 
such agencies are introduced as are incompatible with 
the life of the organisms, such as heat, or treatment 
with strong acids and alkalies. 

Most of the micro-organisms that are of interest to 
us (especially mold-fungi, yeast- or sprouting-fungi, 
and cleft-fungi or schizomycetes) resist these reagents 
strongly; the spirochetes only, which occur in the 
blood during recurrent fever, form an exception since 
they quickly perish in all the different reagents, 
even in distilled water. This capacity of resistance 
of the microbes can also be utilized for their diag- 
nosis, because granules of protoplasm, for instance, 
dissolve in strong acids and alkalies while schizomy- 
cetes remain unchanged. If the latter are grouped 
in so-called colonies (as the zoogloe-masses) they 
often stand out clearly after treatment with strong 
acetic acid or liquor sodse, since the cellular elements 



THE EXAMINATION OF FLUIDS. 121 

and other granular matters, which previously con- 
cealed the colonies, are entirely cleared up. Their 
disposition in chains, or the characteristic form of the 
separate individuals (rod-shaped, oval, etc.), often ren- 
ders the diagnosis of micro-organisms possible with- 
out further difficulty. We must always guard against 
mistakes ; granular unorganized precipitates may be 
taken for micrococci and minute crystals for bacilli, 
and even very small fat-granules may cause a careless 
observer to err. 

Koch's Method of Staining Dried Preparations. — 
Under some circumstances it is certainly by no means 
easy, and is often quite impossible, to arrive at a 
positive opinion regarding the significance of minute 
granules that are contained in any fluid by a simple 
examination, and by the application of the ordinary 
micro-chemical reactions. In these, as well as in all 
difficult cases in general, in fact in every instance 
where it is desirable to make permanent preparations, 
the method of drying and staining is employed, which 
originated essentially with Koch and Ehrlich. 

This depends upon these two facts: 1. If a thin 
layer of fluid is dried rapidly, the forms of the cellu- 
lar elements and schizomycetes are not materially 
altered; 2, the schizomycetes are marked by their 
great affinity for the basic aniline dyes, and may in 
this way be distinguished from other granular bod- 
ies. It should always be observed that not only the 
schizomycetes, but other bodies also (as cell-nuclei 
and their fragments and certain protoplasmic gran- 
ules) show the same affinity for the dyes in question, 
so that in using this method it is necessary to criticise 



122 TEE USE OF THE MICROSCOPE. 

strictly the value of tlie objects found. It is also 
probable tliat there may be varieties of schizomycetes 
which do not possess this affinity ; the forms thus far 
known all show a very strong capacity for dyeing, 
but many, however, stain only under certain fixed 
conditions. The process is as follows: The fluid is 
spread out in the thinnest possible layer upon the 
cover-glass or slide, either by separating the drop into 
a very thin layer by means of a needle or platinum 
wire, or by placing another cover-glass upon it and 
again removing the same. The beginner easily fails 
by making the layers too thick ; they must be as thin 
as those used for the examination of the blood. 
Then the fluid is dried in the air, and is exposed for a 
few minutes to a temperature of 120° C. ; it is enough 
to pass the glass with the dried liquid carefully 
through the flame of a gas-burner three times, with a 
motion about as rapid as that with which one cuts 
bread (Koch). The necessary experience in warm- 
ing the specimen sufficiently, without overheating it, 
is soon attained. Warming is especially required for 
fluids that are rich in albumin, its principal purpose 
being to transform the albumin into an insoluble va- 
riety; it must not be prolonged above five or ten 
minutes when schizomycetes are present, else their 
staining power will be impaired. After warming* 
the preparation is stained. A drop of a strong solu- 
tion of gentian-violet, methyl-blue, fuchsin, or Bis- 

* Heating may be dispensed with in the case of non-albuminous 
fluids ; layers of strongly albuminous fluid, on the contrary, if they are 
simply dried and are then treated with staining- solutions, are apt to swell 
readily and to partially dissolve ; hence with these the heating or co- 
agulation of the albumin precedes. 



THE EXAMINATION OF FLUIDS. 123 

rnarck-brown, in short of any basic aniline dye, is 
poured upon it and is left standing for a little while 
(from one to several minutes), after which it is 
washed off with distilled water; a brown, blue, or 
red cloud is then observed at once upon the glass. 
The examination may be undertaken directly, by 
placing the cover-glass, with the dried and stained 
layer of fluid upon its under side, on the slide with a 
drop of distilled water. The water adhering to the 
upper surface is easily removed by sucking or blow- 
ing it with a glass pipette. If the under surface of 
the cover is again dried (and this is also accomplished 
most quickly by blowing upon it through a tube), 
the specimen may be at once permanently mounted 
in a drop of Canada balsam, prepared with chloro- 
form. If a longer time (several minutes) is desired 
for staining, the coloring of the film of fluid that has 
been dried on the cover may be effected in a watch- 
glass. Most of the other granular elements are also 
stained, but methyl-blue has the advantage, according 
to Ehrlich, of not overstaining even after acting for 
hours. 

The cellular elements may generally be preserved 
perfectly well in their original forms in such a prep- 
aration ; the changes of shape in some of these, caused 
by the spreading out of the fluid (comet-shaped fig- 
ures), are very readily recognized as such. The nuclei 
and also the schizomycetes are stained with special 
intensity, so that in this way they are brought into 
view in a striking manner. Ehrlich has recently 
recommended very highly for staining schizomycetes, 
methyl-blue in a concentrated watery solution that 



124 THE USE OF THE MICROSCOPE. 

must act for half an hour and longer. In the author's 
experience the before-mentioned dyes, especially gen- 
tian-violet, have given about the same results as me- 
thyl-blue. 

Every one who becomes acquainted with this sim- 
ple method must adopt the opinion that these are 
undoubtedly the best ways hitherto known of demon- 
strating micro-organisms in fluids (that is, with the 
modification proposed by Gram. Compare p. 75). 
In examining specimens thus prepared with strong 
immersion-systems and with an open condenser, we 
recognize at once the sharply-defined, deeply-stained 
micro-organisms in their characteristic forms and 
groupings, and learn very soon to diagnosticate as 
such certain impurities or precipitates, and to distin- 
guish them from schizomycetes. In mucous fluids 
principally, such as synovia, we have to contend 
with precipitates, since the mucus always takes a 
pretty deep color with these dyes; nevertheless it 
only requires slight practice in order to show prop- 
erly the irregular, granular, and thready masses. 
By treating the stained specimens for a short time 
with a dilute solution of iodine and iodide of po- 
tassium, the color of the schizomycetes usually ap- 
pears more intense ; or the nuclei may be subse- 
quently decolorized by treatment with alcohol, and 
separate staining of the parasites may be produced 
(Gram). 

Finally, it should be observed that tubercle-bacilli 
are never stained by this method ; they differ in this 
respect from all the other known forms of schizomy- 
cetes. Baumgarten has even proposed to make use 



THE EXAMINATION OF FLUIDS. 125 

of their negative peculiarity, or failure to stain, for 
their rapid recognition in sputa. 

Tissue-fluid can be examined for schizomycetes, 
after being dried and stained, precisely like ordinary 
fluids. If a fresh, clean-cut surface of an organ be 
stroked with a heated platinum wire a sufficient 
quantity of liquid is generally obtained, which is then 
rubbed directly over the cover-glass with the wire. 
This process is very valuable as furnishing the most 
convenient and rapid demonstration of the micrococci 
of pneumonia. 

He who desires to pursue original investigations 
will naturally not be satisfied with the simple demon- 
stration of micro-organisms, but will be obliged to 
study their peculiarities more closely. The method 
of drying upon the cover-glass is very convenient for 
this purpose ; fifty or more dry preparations can be 
very easily made from a single fluid or from a cut 
surface of an organ, and can be kept unstained in a 
small dish as long as is desirable. In this way a large 
number of nearly identical cover-glass preparations 
are obtained, which can be studied in various ways 
by the aid of chemical reagents and different stain- 
ings. 

It is quite clear that all schizomycetes do not re- 
act in the same manner with all the basic aniline 
dyes ; the most important differences, so far as is yet 
known, are those displayed by the encapsulated mi- 
crococci of pneumonia. However, we may assume 
that, with continued investigations, other schizomy- 
cetes will be found to manifest specific reactions of 
this character. 



126 TEE USE OF TEE MICROSCOPE. 

1. Blood. — The examination of blood is easily 
effected, in accordance with the principles already 
stated. A drop of blood, obtained either by copious 
bleeding or by the prick of a needle, is taken up 
neatly and covered ; great care must be employed to 
have the layer extremely thin, so that never more 
than a single stratum of blood-corpuscles may be 
present. When the drop is to be taken from a punct- 
ured wound, the skin in its neighborhood must be 
carefully cleaned and dried, and the needle that is to 
be employed must be heated just before using ; in 
spite of these precautions we should be prepared to 
meet with certain impurities, though only such as 
epidermal scales, etc. It is better to wipe away care- 
fully the drop which first wells up, to allow a new 
one to appear, and then to take up a small portion of 
this on a cover-glass that is held over it ; the cover 
with the adherent drop (which should be at most 
not larger than the head of a small pin) is then placed 
gently on the slide, so that the blood is spread out in 
a very thin layer between the two glasses. 

If the layer has been made sufficiently thin, we at 
once recognize between the discoid red blood -cor- 
puscles the clear transparent plasma and the white 
cells, which, as is known, exist in small numbers 
in normal blood. There are also found in normal 
blood variable numbers of small, irregular granules 
or particles, which have been described as elementary 
granules or products of degeneration. There is still 
a difference of opinion as to their significance. It 
is probable that elements possessing quite different 
degrees of importance are hidden away among them ; 



THE EXAMINATION OF FLUIDS, 127 

the " blood-plates " recently described by Bizzozero, 
which, according to this author bear a close relation 
to coagulation, have always been regarded hitherto 
as indifferent bodies resulting from decomposition. 
Hayem describes them as hsematoblasts, or elements 
out of which the red blood-corpuscles are to be 
formed ; it is highly probable that he is wrong. The 
number and size of these little bodies rarely vary ; 
whether they possess any pathological significance or 
not is still uncertain. They were once regarded by 
Lostorfer and Strieker as characteristic elements of 
syphilitic blood, and were called "syphilis-corpus- 
cles " ; if this view were correct every man would be 
syphilitic. 

As regards the action of the ordinary reagents, it 
is well known that distilled water, as well as acids 
and alkalies, causes the red corpuscles to swell and 
become pale ; the haemoglobin is rapidly discharged 
so that the corpuscles almost entirely disappear. In 
order to preserve them as far as possible in their natu- 
ral form, solutions of a certain strength (so-called in- 
different fluids) must be employed : for example, a 
solution of sodium chloride from three-quarters to 
one per cent. ; concentrated salt-solutions always pre- 
serve the blood-corpuscles, but they occasion essen- 
tial changes in their form and size — that is, they 
cause them to shrivel up. 

It is always advisable in examining abnormal 
blood to observe it directly and when undiluted. 
The following are the most common changes in the 
blood : 

(a) Diminution of the Number of Red Blood-cor- 



128 THE USE OF TEE MICROSCOPE. 

puscles in Ancemia. — It is easy after a little experi- 
ence to establish at once, without the aid of additional 
apparatus, the existence of the more marked degrees 
of this change, by comparing a specimen of the patho- 
logical with one of normal blood prepared in the 
same manner. If more exact determinations are de- 
sired, a blood-counting apparatus is required ; the 
instrument most worthy of recommendation is per- 
haps the one devised by Thoma,* which is constructed 
by the optician Zeiss in Jena. The exact directions 
for using this apparatus will be found in the passage 
cited. While the normal number of red blood-cor- 
puscles is about five million to the cubic millimetre, 
it may in severe cases of anaemia fall to iive hundred 
or even to one hundred and forty-three thousand 
(Quincke) ; it is evident that advanced grades will 
have already been clearly recognized without special 
counting. 

(F) Change in the Size and Shape of the Red 
Blood-corpuscles, or JPoikilocytosis. Nucleated Med 
Blood-corpuscles. — The red corpuscles, as is known, 
all possess normally the same characteristic discoid 
shape, with a depression on both sides ; the center is 
thinner, and is therefore less deeply colored than the 
edge* The size of the normal red blood-corpuscles 
also varies within relatively narrow limits, as any one 
can readily prove to himself. There appear in most 
cases of anaemia, but with especial regularity in so- 
called idiopathic pernicious anaemia, beside normal 
corpuscles numerous irregular and, as a rule, smaller 

* Lyon and Thoma, " Ueber die Methode der Blutkorperzahlung," 
"Virch. Arch.," Bd. 84, S. 31. 



THE EXAMINATION OF FLUIDS. 129 

bodies, containing haemoglobin, called microcytes, and 
sometimes also others which exceed in size the nor- 
mal red disks (megaloblasts of Ehrlich). Nucleated 
blood-corpuscles are observed (though rarely) in the 
direct examination of anaemic blood. Ehrlich has 
found, by examining dried and stained preparations 
of blood (obtained according to the method described 
on page 122, except that the warming occupies more 
time), that nucleated corpuscles may be demonstrated 
in all severe cases of anaemia, whether of traumatic 
or idiopathic origin. He showed this point of differ- 
ence, that in traumatic or secondary anaemia nucleated 
blood-corpuscles are found of the same size as the 
normal red disks (normoblasts), while the large 
forms, or megaloblasts, are characteristic of idiopathic 
anaemia. 

As regards the microcytes and poikilocytes, it 
should be stated that they very probably represent a 
degeneration-product, or a form of disintegration of 
the normal corpuscles ; many analogous forms can be 
found in the blood of a cadaver when examined about 
twenty-four hours after death, and under certain con- 
ditions they may be seen to develop in the specimen 
of blood under one's very eyes (Vulpian). In every 
case in which we desire to observe these objects the 
blood must be examined in a state as little changed 
as possible. Certain forms of microcytes are, how- 
ever, to be regarded as artificial products. 

(<?) Increase in the Number of the White Blood- 
corpuscles. Leucocytosis and Leucaemia. Changes in 
the Granular Protoplasm. — In many affections, espe- 
cially in febrile conditions, the white blood-corpus- 



130 THE USE OF TEE MICROSCOPE. 

cles are increased absolutely and relatively as com- 
pared with the red (leucocytosis of Virchow). The 
proportion of the white to the red corpuscles is nor- 
mally one to three hundred, or even less ; we can 
easily learn from a direct examination of the blood 
(which should be observed in this case also in its 
natural state) to estimate pretty accurately the rela- 
tive increase of the white cells. In leucocytosis, a 
condition that may again retrograde, the white cor- 
puscles are increased to one in fifty, and even more ; 
in leucaemia, which is, as a rule, a persistent and 
necessarily fatal disease, the proportion increases so 
much in the most serious cases that the white cor- 
puscles exceed in number the red. At the same time 
the absolute number of the red disks is very consider- 
ably diminished ; the counting-apparatus is necessary 
for the precise determination of these proportions. 

The condition of the granular protoplasm within 
the leucocytes is a subject of extreme interest ; this 
has been studied of late by Ehrlich.* Ehrlich pre- 
pares dried specimens of blood in very thin layers, 
and heats these for some time at a temperature of 
about 120° C. If different staining-solutions are now 
allowed to act upon these preparations, constant vari- 
ations in the coloring of the protoplasmic granules 
within the leucocytes are obtained, which are of 
great physiological, as well as diagnostic, signifi- 
cance. He distinguishes in this manner five differ- 
ent kinds of granules, from the alpha to the epsilon 

* Ehrlich's statements with reference to his methods of staining and 
their results are scattered throughout several dissertations, written by 
his pupils, as well as in a number of minor articles in different places— 
a very injudicious way of publishing. 



THE EXAMINATION OF FLUIDS. 131 

variety; the a granules, also called eosinophil gran- 
ules, are characterized by their property of staining 
deeply with acid dyes, such as eosin. These eosin- 
ophil granules are present in only a very few normal 
human white corpuscles, and it is possible to distin- 
guish a beginning leucaemia from an ordinary leuco- 
cytosis by the presence of numerous eosinophil cells. 
The demonstration of these cells is, according to Ehr- 
lich, very simple ; a dried and warmed blood-prepa- 
ration having been rapidly stained with a drop of a 
solution of eosin in glycerin, is washed in water, and 
is then dried and mounted in Canada balsam. If the 
eosinophil cells are increased in number, they will be 
seen at once as red bodies. 

(d) Other Cell-Elements which appear in the Blood. 
Worms and Schizomycetes. — During typhoid fever 
there are found in the blood large cells, which con- 
tain in their interior several red blood-corpuscles 
(Eichhorst). It is quite probable that these come 
from the spleen, since at autopsies similar bodies 
are found regularly in the splenic tumor of typhoid. 
Flat cells loaded with fat-drops are frequently ob- 
served in the blood in acute infectious diseases, 
especially in recurrent fever; they are regarded as 
endothelial cells from the walls of the vessels. Cells 
containing granules and flakes of black pigment, as 
well as free masses of the same, appear in the blood 
in severe malarial poisoning (melanaemia). Tumor- 
cells, which circulate in the blood in cases of malig- 
nant metastatic growths, we can scarcely expect to 
find in examining the blood obtained from the capil- 
laries by a needle-puncture or cupping-glass ; these 



132 THE USE OF THE MICROSCOPE. 

cells are only characteristic, as a rule, when they reach 
considerable dimensions, so that they can not pass 
through the narrow capillaries. Of the animal para- 
sites, Filavia sanguinis hominis and Distoma hw?na- 
tobium appear in the blood of man, but both are 
found only in tropical, or sub-tropical, countries. 

Schizomycetes have hitherto been regularly dis- 
covered in human blood only in two affections, the 
Bacillus anthracis in anthrax (Davaine), and the 
Spirochceta Obermeyeri in recurrent fever.* The 
blood is examined directly in a very thin layer, with- 
out the addition of any reagent, or dry preparations 
may be heated in the manner already described, and 
stained with gentian-violet, methyl-blue, etc. The 
anthrax-bacilli are slender, motionless rods which re- 
sist most reagents ; the spirilli of recurrent fever, on 
the contrary, move very actively, and are easily de- 
stroyed by most fluids that are added to them, even 
by distilled water. The presence of the spirilli is, as 
is known, limited to the febrile stage of the disease; 
they are found only very rarely for a short time after 
the attack. They are, on the other hand, never ab- 
sent during the progress of the fever, so that they 
may be regarded as a sure diagnostic criterion of this 
affection. However, they are sometimes present only 
in small numbers, even in rather severe cases, and may 
perhaps be overlooked on a cursory examination. It 
is advisable in these instances, if it appears to be 
of sufficient diagnostic importance, to remove several 
grammes of blood by means of a cupping-glass, and 

* Tubercle-bacilli have as yet been found by Weichselbauni only in 
the blood of a subject after death from general miliary tuberculosis. 



THE EXAMINATION OF FLUIDS. 133 

to allow it to coagulate. The spirilli are then apt to 
gradually collect at the edges of the clot, to the num- 
ber of twenty or more, and are even rolled into a 
knot, or united together in the form of a rat-king 
(Rattenkonig*). Since, after being removed from 
the body,f they continue their active lashing move- 
ments for hours, or even days (although kept at the 
temperature of the room), when collected in groups 
they cause violent currents in the fluid, so that at- 
tention is often directed to them even when a low 
power is used. The spirilli may be very easily stained 
in dry preparations with the different aniline dyes, as 
gentian-violet. The history of their development is 
still unknown; whether the supposed movable cor- 
puscles and double granules, which have been seen in 
the blood in recurrent fever, as well as in other infec- 
tious diseases, and sometimes even in normal blood, 
and have been described as " micrococci," or spores, 
are really to be regarded as such, is still doubtful. 

In every case the reports concerning the occur- 
rence of micro-organisms in normal blood are thor- 
oughly untrustworthy; most of the communications 
also concerning the so-called micrococci, monads, or 
rods, seen in the blood during various infectious dis- 
eases, such as diphtheria, hospital gangrene, erysipe- 
las, etc., as well as in intermittent fever, are not suffi- 
ciently supported. Even in pyemia and in ulcera- 
tive endocarditis no organisms can usually be de- 
monstrated by an examination of the blood of the 

* This expression signifies several rats, which have heen attached 
together hy their tails. — Teans. 

t It has heen recently stated that they are even ohserved to in- 
crease in number outside of the body (Albrecht). 



134 THE USE OF THE MICROSCOPE. 

living, while after death the capillaries in many 
places are found to be plugged with micrococci. It 
is not unlikely that in such cases the organisms enter 
the blood in batches, possibly only in small numbers, 
and that they then soon lodge in the capillaries, in 
which, under certain conditions, they may rapidly 
multiply. It should be remarked also that, although 
micro-organisms are often observed in pus-corpuscles, 
they have, as yet, never been certainly found in the 
interior of the human white blood-corpuscles, although 
the latter are accustomed to appropriate with avidity 
other finely granular matters. Probably the future 
will bring further discoveries in this direction ; it is 
quite possible that within the corpuscle, which we have 
hitherto been forced to describe in a loose way as a 
product of degeneration, there lie concealed important 
forms, which we shall one day learn to differentiate. 

(e) The Examination of Blood- Stains. Ucemin- 
Crystals. Hoematoidin. — Blood, when dried upon 
wood, linen, metallic instruments, etc., is frequently 
the subject of medico-legal examinations. It is very 
often possible, after soaking in proper fluids, espe- 
cially in salt solution (eight tenths of one per cent.) 
and liquor potassae (thirty-three per cent.), to isolate 
blood-corpuscles from such stains, and even their 
shape and size are to some extent preserved. 

Since now the blood of man and of mammalians 
is, as is well known, characterized by the circular 
form of the red blood-corpuscles, while the other 
vertebrates have oval corpuscles, we can generally de- 
cide with perfect certainty, even in the case of dried 
blood, whether it comes from a mammalian (includ- 



THE EXAMINATION OF FLUIDS. 135 

ing man) or from some bird, etc. But beyond this 
our art does not extend ; it is impossible to positive- 
ly identify given blood-corpuscles as human. Most 
mammalians, however, possess rather smaller corpus- 
cles than man ; those from the blood of the sheep and 
goat are on an average only a little over half as large 
as human corpuscles, but other animals, as the dog 
for example, resemble man very closely in this re- 
spect. It is better in every instance, when deciding 
upon blood-stains, merely to state in a given case 
that the blood came from a mammalian ; it is impos- 
sible to assign a positive value to the relations of 
size, because of the differences in the mode of drying, 
and of swelling when fluid is added, which vary 
with the age of the spot, etc. The so-called Teich- 
mann's hsemin-crystals are also obtained from dried 
blood. These consist of hydrochlorate of haematin, 
and are prepared in the following manner: To a 
small quantity of dried blood placed upon a slide 
(for example, a thread dipped in blood) add a few 
drops of glacial acetic acid and a particle of salt; 
then warm the slide gradually until bubbles begin to 
form. There will be observed, forming around the 
thread, a large number of dark brown rhombic crys- 
tals, which are perfectly insoluble in water, and show 
exquisite double refraction. This test furnishes posi- 
tive results even in the case of quite old blood-spots ; 
it is self-evident that this process can be applied to 
every blood-stained object. It is frequently necessary 
to first soak out the suspicious spot in water, and 
then to apply further tests to the watery extract. 
From these artificial products are to be distin- 



136 THE USE OF THE MICROSCOPE. 

guished the haematoidin crystals, which also occur in 
a rhombic shape in old blood-extravasations, in the 
corpora lutea of the ovaries, etc., partly free and 
partly inclosed in the interior of cells. They are of 
a bright ruby or orange-red color, but contain no iron ; 
they are soluble in chloroform and resemble bilirubin 
very closely, in fact they are regarded by many as 
identical with bilirubin. 

Hsonioglobin-crystals, so far as is now known, do 
not appear normally in man, while these and other 
bodies have been found in large numbers in the puer- 
peral uterus of the Guinea-pig ; they may be prepared 
in different ways, for instance, by the action of a 
concentrated solution of pyrogallic acid upon blood 
that has been previously diluted with distilled 
water. 

2. Sputa. — The microscopical examination of 
sputa is of extreme diagnostic importance, and is 
therefore very frequently undertaken. The technique 
of this examination is quite simple : it is first deter- 
mined by gross inspection what substances can be 
distinguished in the sputum, since the latter always 
presents a number of different kinds of material 
which arise from various sources. Particular atten- 
tion must be paid to opaque white, or grayish- white, 
plugs, which are best seen if the sputum is spread 
in a thin layer upon a polished black porcelain 
plate; in these plugs we generally find at once 
elastic fibers, which should be preserved, since they 
are to be regarded as cast-off pieces from the wall of 
a cavity. The so-called " asthma-crystals " of Leyden 
are also found as a rule in the interior of greenish- 



TEE EXAMINATION OF FLUIDS. 137 

wliite miliary plugs, which may be distinguished, even 
by the naked eye, in the otherwise clear sputum. If 
also we have occasion to look for echinococci, or for 
other rarer additions, in short in every case, a careful 
macroscopical examination should first be made ; a 
neglect of this caution often leads to errors and 
negative results. Microscopical preparations are then 
made of all these different portions, which are fre- 
quently arranged in layers one above the other, by 
transferring a bit of mucoid material to a slide, by 
means of a needle and spatula, and covering it with a 
cover-glass. It is seldom necessary to employ a dilut- 
ing fluid, such as salt-solution or distilled water. In 
examining microscopically, begin with a low power 
(from fifty to eighty), and only change this for 
stronger lenses after yon have studied the entire 
specimen with the former. The elastic fibers are 
usually recognizable to some extent, even with a 
weak lens, either directly, or by the dark, friable ma- 
terial in which they are imbedded; since the low 
power furnishes a correspondingly larger field of 
vision, there are more chances of finding them with 
this than with a high power. Besides, the former 
is not so much affected by slight differences of focus, 
so that a number of superposed layers in the speci- 
men may be examined simultaneously. 

Those portions of the preparation which are rec- 
ognized by the low power as being peculiar, are then 
analyzed more closely with the high objective ; then 
only is the microscopical diagnosis established. 

(a) Oral Fluids. — Impurities of the most varied 
description are almost always encountered during the 



138 THE USE OF THE MICROSCOPE. 

examination of sputa. We need never expect to ob- 
tain the contents of the bronchi in an unmixed state ; 
the secretions of the niouth, salivary glands and throat 
at least are mingled with it. We must accordingly 
learn to recognize these perfectly. Large numbers 
of epithelial cells from the mucous membrane of the 
mouth and throat are always found normally in the 
oral fluids. You will very soon learn to recognize 
these large, irregular plates which, when treated with 
acids or alkalies, swell up to form globular vesicles, 
and are generally filled with numerous schizomycetes. 
In catarrhal conditions of the mucous membrane of 
the mouth and throat there also appear in the oral 
fluid living epithelia, which throw out knobbed pro- 
cesses and execute feeble amoeboid movements. 

In oral catarrh the superficial layers of epithelial 
cells are often cast off en masse, and in the coating on 
the tongues of individuals thus affected we find in 
very large numbers the goblet-shaped tips of the fili- 
form papillae, which consist of resistant, horny epithe- 
lial cells, closely adherent to one another. 

We find also many round cells, mucous or sali- 
vary corpuscles, which come from the mucous or sali- 
vary glands (especially from the submaxillary and 
sublingual), and to a less extent also from the deeper 
layers of the stratified pavement epithelium. These 
are originally small amceboid cells, analogous to 
lymph-corpuscles, but under the influence of the di- 
lute parotid saliva they become changed, assuming a 
globular shape, while a limiting membrane stands out 
in contrast with the clear contents ; the latter in- 
cludes one or two round nuclei, besides a large num- 



THE EXAMINATION OF FLUIDS. 139 

ber of fine granules, wliicli are always seen in active 
dancing motion — molecular motion in salivary cor- 
puscles. These granules are not, as lias been sup- 
posed, parasitic organisms, at least they never stain 
in aniline dyes; whether their active movement 
represents a vital phenomenon or not, somewhat 
analogous to the protoplasmic currents in the cells of 
plants, has not been determined. 

But, besides these, numbers of micro-organisms of 
different sorts roll about freely in the fluids of the 
mouth, such as stiff, long threads of leptothrix of 
varying breadths; globular cocci, of different sizes, 
frequently arranged in the form of chains or compact 
groups, and not seldom bacilli; and very beauti- 
ful forms of spirochaetes, which in their shape and 
serpentine movement closely resemble the spirilli of 
recurrent fever, except that they usually attain much 
larger dimensions than the latter. The less careful a 
person is about cleansing his mouth, the larger are 
apt to be the masses of micro-organisms which collect 
there ; but they are never entirely absent, even when 
the greatest cleanliness is observed, for the germs of 
schizomycetes, that are always contained in the in- 
spired air, invariably find in the fluids of the oral 
cavity the most favorable nidus. One species of these 
was regarded as the cause of dental caries, but this 
is probably erroneous (W. Miller). By inoculating 
animals it is found that there are a great many 
pathogenic cleft-fungi in the normal human saliva. 

Budding fungi are only occasionally present in 
large numbers in the oral fluids, but a species of 
thready fungus, the parasite of aphthae, or oidium 



140 THE USE OF THE MICROSCOPE. 

albicans, is frequently found ; this occurs, as is known, 
most often in bottle-fed children, and in adults whose 
systems are greatly reduced, as in the phthisical. 
This fungus consists of branched or limb-like threads 
with oval spores, which form a more or less thick 
mycelium between the epithelial cells of the super- 
ficial layers and upon the surface itself. 

There often appear in the saliva, in addition to 
these elements, various remnants of vegetable food. 
For several hours after a meal many persons retain 
in their mouths, especially between the teeth or in 
carious cavities of the same, microscopical sam- 
ples of their repast, such as often astonish and per- 
plex the beginner when examining the expectora- 
tion. 

The mucous fluid contained within the nasal cavi- 
ty, with which blood is frequently mixed, may also 
occur in the expectoration as an impurity, as w x ell as 
the contents of abscesses that open into the cavity of 
the mouth and throat (dental, alveolar, tonsillar, and 
retropharyngeal abscesses). Special attention should 
be called in this connection to the small concrements, 
sometimes exceeding a pea in size, which often form 
in the crypts of the tonsils, through the incrustation 
of the retained secretion with lime, and are occasion- 
ally dislodged during a fit of coughing. The patient, 
and often the physician also, are greatly disturbed by 
the appearance of this supposed pulmonary calculus, 
but the correct diagnosis is very easily made on mi- 
croscopical examinations. The lime is dissolved by 
the addition of dilute hydrochloric acid, and there re- 
main then the large, horny, epithelial cells (which are 



THE EXAMINATION OF FLUIDS. 141 

sometimes disposed in the form of concentric balls) 
and numerous micro-organisms. 

(b) Products of 'the Respiratory 'Mucous Membrane. 
— After you have made yourself perfectly familiar 
with all these elements which appear accidentally in 
the expectoration, you can proceed with profit to the 
microscopical examination of the latter. The chief 
component of the expectoration is the excretion of 
the inflamed mucous membrane of the respiratory 
tract, a fluid containing more or less numerous round 
cells. The secretion of the mucous membrane of 
the throat and upper part of the larynx, as far as 
this is lined with stratified pavement epithelium, is 
also exceedingly rich in cast-off cells ; in the upper 
part of the respiratory tract, on the contrary, as 
well as in the lower portion of the larynx, trachea 
and bronchi, and at every point where the membrane 
is covered with long, cylindrical ciliated epithelium, 
desquamation occurs only in exceptional cases. It is 
rare to meet with ciliated cells, or their remains, dur- 
ing the examination of sputa. 

The vitreous, transparent portion of sputa con- 
tains but few formed elements ; the more numerous 
the latter, so much the more turbid and opaque is the 
sputum. 

Round cells occur most frequently in purulent ex- 
pectoration, in which case there are generally num- 
bers of fine fat-granules in the protoplasm of the cells, 
which increase their opacity, and give them a yellow- 
ish tint. The round cells are, in most instances, dead 
and motionless, and consist of protoplasm containing 
dark granules and one or more nuclei ; a special wall 



142 THE USE OF THE MICROSCOPE. 

is not usually present, but the edge of tlie cell pre- 
sents fine indentations winch look as if they had been 
gnawed out. The protoplasm is usually so extremely 
granular that the nucleus (or nuclei) is concealed ; the 
latter comes out more clearly after being treated with 
acetic acid, while the granules of protoplasm for the 
most part disappear. The majority of the round cells 
that are found in the sputum are about the size of 
white blood-corpuscles, or a little larger. It may, 
in fact, be assumed that a large part of them are to 
be regarded at once as emigrated white blood-corpus- 
cles, while another part may originate from the mu- 
cous glands, or from the inflamed mucous membrane. 
But in addition to these we find not infrequently 
larger round cells of an epithelial type, which are 
characterized by thin, clear, somewhat circular out- 
lines and single vesicular nuclei ; that is, the nucleus 
is bordered by a dark, sharply-drawn line, while its 
interior is clear and contains one or more nucleoli. If 
these epithelioid cells are stained (which is best accom- 
plished by treating the dried and heated preparation 
with basic aniline dyes) the nuclei do not take such a 
dark and even shade as those of the ordinary small 
round cells, but a difference may always be recognized 
between the deeply- stained periphery and the clear 
center. A few fat-granules are also found now and 
then in the protoplasm of these larger cells, and often 
also black pigment, in the shape of minute granules or 
irregular scales — so-called lung-pigment ; blackish, or 
coarsely-gray spots and streaks are frequently ob- 
served, even on macroscopic examination. By micro- 
scopical analysis it is proved that the black staining is 



THE EXAMINATION OF FLUIDS. ' 143 

hardly due to the abundance of pigmented cells, and 
that free pigment is usually present in only a small 
amount. These dark granules and scales in the cells 
are all to be regarded as inhaled coal-dust that has 
fallen upon the surface of the respiratory mucous 
membrane, has been retained there, and has been sub- 
sequently taken up into the interiors of the amoeboid 
cells. These pigmented cells are almost never absent 
from the secretion of the throat and respiratory tract 
in the case of individuals who have ample opportunity 
to inhale coal-dust, such as miners and inveterate smok- 
ers ; but every man who, under our conditions of civil- 
ization, lives for the most part in closed and heated 
apartments, or even in the open air exposed to the 
influence of sooty chimneys, has an opportunity with 
every breath that he draws to introduce finely-divided 
coal-dust into his respiratory organs. The more mu- 
cus there is secreted on the surface of the respiratory 
tract, the more coal-dust there is retained ; a portion 
of it makes its way into the interstitial tissue of the 
lungs and into the lymph-glands, through the aid of 
the amoeboid cells, while another part is cast off again 
with the sputum. 

Genuine pigment, however, or that which arises 
within the organism itself, appears very seldom; it 
is at once recognized by its brownish (not black) 
color and it points either to a previous haemorrhage, 
or to an obstruction in the venous circulation of the 
lungs, with the escape of red blood-corpuscles and 
the transformation of the same into pigment (brown 
induration of the lungs). Hsematoidin-crystals are 
sometimes found also. In the larger epithelioid cells 



144: TEE USE OF TEE MICROSCOPE. 

of the sputum which have been referred to there 
are often seen collections of homogeneous, glistening 
granules, which, purely on account of their external 
resemblance, are described as " my elin-granules " ; 
nothing certain is known regarding their nature and 
significance. In Buhl's well-known demonstrations * 
particular stress was laid upon these larger round 
cells in sputum ; they were considered to be desqua- 
mated epithelial cells from the alveoli of the lungs, and 
because of their occurrence in great numbers in the 
expectoration the diagnosis of "desquamative pneu- 
monia," that is of incipient phthisis, was made. These 
views have been shown to be untenable ; large num- 
bers of these cells, even gathered together in masses, 
and filled with granules of " myelin," fat and pigment, 
have been found in the morning sputum of entirely 
healthy men, and in simple bronchial catarrh, f It 
is likewise extremely doubtful if they were really re- 
garded in all cases as cast-off epithelial cells of the 
pulmonary alveoli; on the contrary it has become 
probable as the result of many examinations that 
under some circumstances ordinary migratory cells 
(leucocytes, etc.) may be transformed into such epithe- 
lioid elements. 

Among other cellular forms, we occasionally en- 
counter cells filled with fat-granules (in sub-acute 
pneumonic processes), and rarely giant-cells (in tuber- 
cular phthisis). 

* Buhl, " Lungenentzundung, Schwindsucht, tmd Tuberculose," 
Mlinchen, 1872. 

t Compare Guttmann mid Smidt, "Zeitschr. fur klin. Med.," Bd. 3. 
Panizza, "Deutsch. Arch, fur klin. Med.," 1881. Bizzozero, "Cen- 
tralbl. fur klin. Med.," 1881, S. 529. 



THE EXAMINATION OF FLUIDS. 145 

(6) Elastic Fibers. Fibrinous Exudation. 
Asthma- Crystals. — Tlie finding of elastic fibers in 
the sputum is naturally of very great importance, 
since through these we infer at once a degenerative 
process in the interior of the lungs. As already 
stated, we find elastic fibers in the opaque plugs be- 
fore described ; it is frequently possible, by the addi- 
tion of strong acetic acid, to render even very thick 
portions of these plugs quite transparent, so that the 
elastic fibers within them, which are known to resist 
the acid, appear most beautifully. The beginner 
sometimes regards as suspicious small bits of cotton- 
fibers, etc., which also resist acetic acid ; needle-shaped 
crystals of fatty acid which often appear in great 
quantities in sputa during putrid bronchitis, pulmona- 
ry gangrene and similar affections, may possibly, by 
reason of their long, wavy shapes, give rise to error. 
These melt when heated gently and are transformed 
into small fat-granules. We should lay it down as a 
rule to make the diagnosis of elastic fibers only when 
several of these are united and, by their characteristic 
wavy course, make it clear that the framework of an 
alveolar wall is present. 

We frequently meet with larger coherent pieces 
of the framework of the lung, including several alve- 
oli ; the more numerous and the larger these micro- 
scopical fragments of lung are, so much the more seri- 
ous and rapid must we consider the destructive pro- 
cess. 

It should be remarked also that in pulmonary 
gangrene we sometimes fail to find any elastic fibers 
in such expectorated fragments of lung as are 



148 TEE USE OF TEE MICROSCOPE. 

recognizable under the microscope ; these are gradu- 
ally dissolved in the putrid fluids. Traube, who 
called attention to this condition, recognized in this 
a point of differential diagnosis from pulmonary ab- 
scess, in which affection the elastic fibers are preserved 
longer. Even in ordinary caseation of the lungs the 
elastic fibers disappear, though very gradually. 

Fibrinous masses also appear in sputa under cer- 
tain conditions — even coarse fibrinous molds of the 
bronchi in croupous bronchitis, and of the bronchioles 
in acute pneumonia (Reniak). The branched, dichot- 
omous masses of fibrin, which are already recogniz- 
able by the naked eye, show under the microscope 
the familiar appearance of a net-work of delicate 
fibers, which swell up and disappear in acetic acid, in 
which are imbedded numerous round cells and quan- 
tities of micrococci. A brief reference has already 
been made to asthma-crystals; these are pointed 
octahedra, occurring in large numbers (within the 
plugs already described) in the sputum of asthmatics 
during an attack, while they are generally absent in 
the interval. They are occasionally found also in 
non-asthmatic sputum, so that they are not pathogno- 
monic of this disease. They are perfectly analogous 
to the crystals which have been found in the semen, 
in the marrow of bones, in the blood (especially in 
leucaemia), and in various other places, and which, 
according to Schreiner, consist of the phosphate of 
an organized base. 

(d) ScMzomycetes. Tubercle-bacilli. Micrococci 
of Pneumonia. — Schizomycetes appear in large num- 
bers in the sputum because of its mixture with the 



THE EXAMINATION OF FLUIDS. 147 

oral fluids ; in addition to these tliere are forms that 
come from the respiratory organs themselves, as in 
putrid and diphtheritic bronchitis, in purulent catarrh 
of the bronchi and trachea, etc. We are, however, 
not yet in a condition to find our way amid the crowd 
of different forms ; the supposed " discoveries " of the 
fungus of whooping-cough and measles, the micro- 
cocci of diphtheria, etc., have never possessed the 
slightest significance to experts in this department. 
On the other hand, the discovery of tubercle-bacilli 
in the sputum by Koch is of the highest importance. 
For it has been established by the use of Ehrlich's 
method of staining that specific bacilli are rarely 
absent from the sputum of phthisical patients, and that, 
on the other hand, their presence furnishes an abso- 
lutely safe criterion of tubercular affections. The 
staining process, which at once shows the bacilli 
and proves their specific character, is the same 
that was previously described for tubercle-bacilli 
in sections. The dried and warmed preparation 
of sputum is stained for twenty-four hours on the 
cover-glass with a concentrated solution of gen- 
tian-violet or fuchsin in aniline- water (that is, a satu- 
rated, filtered solution of aniline) ; if the staining is 
carried on at a high temperature only a short time is 
necessary. Decolorization is then effected by means 
of strong mineral acids, as twenty-per-cent. hydro- 
chloric, or better, in alcohol to which three per cent, 
of nitric acid is added. All the other schizomycetes 
of the sputum which were originally stained at the 
same time are then decolorized, as well as the masses 
of mucus, etc., but the tubercle-bacilli alone appear as 



148 THE USE OF THE MICROSCOPE. 

deeply-stained rods. We generally meet with them 
in the sputum in a condition of active spore-formation ; 
the spores appear as bright globules which include 
the entire breadth of the bacillus. If there are sev- 
eral spores in a single bacillus, the latter may on this 
account appear to be broken up into a row of granules. 
In order to bring out the contrast better we can sub- 
sequently, by any desired method of staining, add a 
ground color, that must naturally possess a shade 
which offers the greatest possible contrast to the 
stained tubercle-bacilli ; I find that this double stain- 
ing is unnecessary in most cases. 

The Importance of the Discovery of Tubercle- 
bacilli. Severe and Light Cases of Pulmonary 
Phthisis. — It is desirable to add a few observations 
concerning the diagnostic and prognostic importance 
of the discovery of tubercle-bacilli in the sputum, in 
which a short excursion into the field of the gen- 
eral pathology of phthisis can not be avoided. 

Pulmonary phthisis belongs to the province of 
tuberculosis. The separation of cheesy pneumonia and 
bronchitis from the real tubercular form of phthisis, 
as insisted upon by Bernhardt and Virchow, must 
naturally be regarded as correct from the stand-point 
of descriptive anatomy, but the radical division of 
these processes, simply on the ground of their histo- 
logical relations, must be abandoned, especially from 
an etiological and practical point of view. More ac- 
curate histological examinations showed that caseous 
pneumonia and bronchitis possessed the same ele- 
mentary structure as so - called genuine tubercles. 
The author demonstrated this positively in the year 



THE EXAMINATION OF FLUIDS. 149 

1873, in a lecture on "Local Tuberculosis" (Volk- 
mann's " Samnilung klin. Vortrage "). In every case 
of phthisis, even in the forms that are apparently non- 
tuberculoiiSj we always find the characteristic submilir 
ary, noiwascular nodules (containing giant cells), 
which represent the chief type of tubercle after it has 
attained its full development. These facts, hither- 
to unknown, of course limited very essentially Vir- 
chow's doctrine concerning the duality of phthisis, 
which had already encountered serious difficulties; 
the occurrence of a tuberculous pleurisy, for exam- 
ple, frequently observed during the course of a cheesy 
pneumonia, must thenceforth be regarded no longer 
as the invasion of a new disease (as Virchow and 
his school taught), but as little more than the exten- 
sion to the pleura of the same pathological process 
which had already taken place in the lungs. 

The simplicity and clearness of this idea as com- 
pared with the earlier doctrine are at once apparent, 
yet it was a long time before it obtained recognition. 
Several years later Charcot and his school, reasoning 
from the same observations, defended the " unite de 
la phthisie " in a series of works ; Eindfleisch took a 
similar position. Through the later experiments of 
Tappeiner, Cohnheim, Salomonsen, and others, which 
were founded upon Villemin's discovery of the inocu- 
lability of tuberculosis, and especially through Koch's 
remarkable discoveries, it was proved with the great- 
est certainty that phthisis and tuberculosis depend 
upon one and the same etiological factor; at the 
same time it is not denied that, under certain con- 
ditions, other elements may play a part in them. 



150 THE USE OF TEE MICROSCOPE. 

Pulmonary phthisis is in fact to be regarded in the 
majority of cases as a local tuberculosis of the lungs. 
If then, we find in the sputum that parasite which 
we know to be the cause of tuberculosis, the positive 
conclusion to be drawn from this is that a tubercu- 
lar process is present in the respiratory tract, in- 
cluding the mucous membrane of the mouth and 
throat. 

Shall we therefore draw the further conclusion 
that the patient in question has developed general 
tuberculosis, and is in consequence beyond hope? 
No, that would be a gross error. The bacillus of tuber- 
culosis is not only found in those instances in which 
the disease advances more or less rapidly, and eventu- 
ally spreads over the whole system by extension 
to the blood and lymph-vessels, but also in cases 
which remain localized for a long time (for years, or 
even for decades) and finally may become com- 
pletely healed, as well as in cases of " local tubercu- 
losis." Man differs essentially in this respect from 
rabbits and Guinea-pigs, which are principally used in 
experiments ; if a tuberculous affection occurs any- 
where in these animals, in the eye for example, after 
inoculation of the anterior chamber, the tuberculosis 
seems without exception to spread throughout the 
various organs in the course of a few weeks or 
months; as a rule the animals rapidly succumb 
to the disease. Dogs appear to act differently ; 
but as yet few facts have been presented on this sub- 
ject. On the contrary, it is perfectly certain that in 
man the affection caused by the tubercle-bacilli may 
in many cases remain for several years comparatively 



THE EXAMINATION OF FLUIDS. 151 

benign and localized, or may become more or less 
completely cured ; however, as long as the process 
continues, the danger is always present that it may 
suddenly increase in intensity and, without our 
being able to demonstrate the causative agents, may 
extend rapidly, either in a local or general manner. 
The human organism seems to furnish in most in- 
stances only a moderately favorable field for tubercle- 
bacilli, so that they generally multiply very sparing- 
ly; if under certain conditions, which are still but 
little known, a rapid development of the parasites 
takes place, this causes a speedy advance in the pro- 
cess. Unfortunately we are not able at present to 
control those conditions through which the increase 
of the microbe is frequently retarded or prevented ; 
if we could attain this end, we should possess at 
once the therapeutics of tuberculosis. We should 
cherish the hope that even this lofty goal is not 
unattainable. At any rate, the paths which lead to it 
have been rendered smooth. If, too, we find tubercle- 
bacilli in the sputum, we infer from these the pres- 
ence of a tuberculous process which, by reason of ex- 
tensive, or rapid local, disturbances and the invasion 
of other organs, may be extremely dangerous; but 
the possibility of a very slow, harmless course, and 
even of healing, is likewise present. 

By the discovery of tubercle-bacilli the diagnosis 
of a tuberculous process in the lungs is now rendered 
possible in many cases in which it was previously 
impracticable or very difficult. The bacilli occur in 
great numbers upon the walls of even the most mi- 
nute phthisical cavities, in tuberculous ulcers of the 



152 THE USE OF THE MICROSCOPE. 

bronchi, etc., and on account of their remarkably 
sharp outlines, through their capacity for isolated 
staining, they are found much more easily and 
quickly in the sputum than the elastic fibers, the 
presence of which formerly constituted the only 
proof of a destructive process in the lungs. By care- 
ful examinations of sputa, therefore, we shall rec- 
ognize as phthisis those extremely numerous light 
and favorable cases which were formerly regarded 
as " suspicious " pulmonary catarrhs, bronchitis, etc., 
just such cases, in fact, as cause insignificant sub- 
jective troubles. And how very frequent such 
favorable cases of "phthisis" are, we learn best 
at the post-mortem table. In nearly one-half of 
the cases of healthy, powerful adults, who have 
died as the result of accident or acute disease, there 
are found on careful inspection traces of destruc- 
tive phthisical processes in the lungs, in the form of 
caseous masses, often incrusted with lime, and cavities 
surrounded by firm, slaty and cicatricial tissue. Many 
of these cases have been entirely, or almost entirely, 
latent throughout their course ; at any rate, in the 
majority of them the suspicion of a serious pulmonary 
affection has never been aroused; in every instance, 
however, tubercle-bacilli could at some time have 
been demonstrated in the sputum. It is well known 
to every practitioner, and has been frequently con- 
firmed by clinical and anatomical facts, that a tuber- 
culous pleuritis, or even a fatal tuberculous meningi- 
tis, may suddenly develop from phthisical centers 
which either appear to have healed perfectly, or have 
been entirely latent, and which need not be very 



THE EXAMINATION OF FLUIDS. 153 

extensive. Whenever tubercle-bacilli are found in 
the sputum, the prognosis given should be a serious, 
but not necessarily a bad one, unless there is addition- 
al evidence. It is certainly known that extensive 
phthisical processes in the lungs are arrested under 
favorable circumstances, and that every incipient 
phthisis need not go on to destruction of the organs. 
However, the diagnosis of tuberculous disease will al- 
ways have a positive influence upon the regimen of 
the patient. It is highly probable that, through the 
early recognition of tuberculosis, the lives of many 
patients can be preserved, by placing them under such 
climatic conditions as have been proved by experience 
to exercise a favorable influence upon incipient 
phthisis. 

Further investigations alone must teach how far 
we can decide concerning the extent of the phthisi- 
cal process from the number of bacilli that occur in 
the sputum. 

On the other hand, the absence of tubercle-bacilli 
in the sputum, if this is constant, is to be regarded as 
a sure sign that destructive tuberculous processes are 
not present in the lungs at the time. If elastic fibers 
occur in the sputum, while at the same time the tu- 
bercle-bacilli are absent, some other degenerative 
process must be inferred, such as the formation of an 
abscess, the breaking down of a tumor, etc. It should 
also be observed that there are certain chronic ulcera- 
tive conditions of the lungs which are not of a tuber- 
culous character, and in which bacilli are not found ; 
these cases are extremely rare. Such observations 
have been made by Biegel in diabetic lesions of the 



154 THE USE OF THE MICROSCOPE. 

lungs, but, as a rule numerous bacilli are present in 
diabetic phthisis. The anatomical appearances of 
lungs from which, the parasites are absent may re- 
semble closely those of ordinary tuberculous phthisis. 

It is self-evident that one should approach the 
examination of the sputum for tubercle-bacilli only 
when he is provided with the best aids. In favora- 
ble preparations, however, they may be recognized 
even with rather low powers; and if they are 
numerous, they may sometimes be distinguished at 
once with the naked eye through the difference in 
staining. But it would be entirely a mistake to at- 
tempt to search for tubercle-bacilli with any except 
the best immersion-lenses, for it is quite possible, ac- 
cording to the author's experience, that bacilli which 
are present in the specimen and were not found with 
weak systems (dry lenses, for example), were over- 
looked, and appear clearly only under corresponding- 
ly higher powers — at least six hundred. It is never 
possible to give a negative opinion with reference to 
tubercle-bacilli without the use of strong, excellent 
immersion-lenses (preferably oil-immersions), and of 
course Abbe's apparatus. He who dreads the ex- 
pense necessary in providing these rather expensive 
aids, or who shrinks from the trouble which is re- 
quired in working with them, must renounce the idea 
of examining schizomycetes. 

Micrococci of JPnemonia. — It is known that a pe- 
culiar structure, or capsule, can be demonstrated in 
the micrococci of acute croupous pneumonia, which 
comes out with especial clearness on staining the dry 
preparation with gentian- violet or fuchsin. The cap- 



THE EXAMINATION OF FLUIDS. 155 

sule is in this way colored less deeply than the coccns 
itself, and usually presents a sharp outline at its outer 
edge. It is still doubtful if this peculiar formation 
can be made use of for purposes of diagnosis ; several 
observers have already obtained results in this direc- 
tion which seem to be positive. But encapsulated 
micrococci are sometimes seen in the sputum when 
no pneumonia is present, so that caution is advised. 
There frequently appear around micrococci colorless 
spaces, which have very little significance, while the 
pneumococci possess capsules that may be stained. 

3. Pus.— Pus consists in general of a fluid (serum) 
in which are suspended small round cells, the pus-cor- 
puscles; it also contains micro-organisms in most 
cases. 

(a) Pus- Corpuscles and Fatty Degenerated Cells. 
Pus-corpuscles are very similar to, or identical with, 
the white corpuscles of blood and lymph. If exam- 
ined when fresh, that is, in pus which has only been 
evacuated for a short time, thev show amoeboid move- 
ments, and then possess the characteristic glistening 
appearance of living protoplasm. In the majority of 
-instances, however, they are already dead and their 
protoplasm has then become coagulated in the form 
of coarse granules ; the nucleus (or nuclei) is usually 
concealed. Many very fine fat-granules are seen 
scattered throughout the protoplasm, and these are 
abundant in such pus-corpuscles as have been dead 
for some time, especially in the contents of so-called 
cold abscesses. 

Pus-corpuscles are generally of nearly equal size ; 
that is, about as large as medium-sized white blood- 



156 THE USE OF THE MICROSCOPE. 

cells. But larger cells, which usually have vesicular 
nuclei, are frequently mingled with them ; if numbers 
of fat-granules are collected in these, the well-known 
fat-granule cells are formed. The latter, also, are 
occasionally alive and perform amoeboid movements. 
They are recognized with a weak lens as rather large, 
dark masses between the ordinary pus-cells; their 
dark outline, when viewed with low powers, induces 
the beginner not infrequently to think of pigmenta- 
tion, although it is entirely due to the many super- 
posed fat-granules. 

The light which comes from below is, by reason 
of its many transitions from the fluid into the fat- 
drops, and from the latter back to the fluid or proto- 
plasmic substance, reflected as if from the surface of 
spherical mirrors, so that it does not reach the eye of 
the beholder ; hence the impression of black. 

The same fatty cell, if illuminated from above, 
appears of a clear white color, likewise in consequence 
of its numerous reflecting surfaces ; if, then, the light 
coming from the mirror is shut off, the fat-cells 
look like glistening white spheres in the dark field, 
it being assumed that the light from above can gen- 
erally impinge upon the preparation. Strong object- 
ives must usually be approached so close to the 
specimen that it is completely shaded. However, 
there are also real pigment-granules, which are mostly 
inclosed in cells, often in pus, as the remains of blood- 
extravasations ; they are distinguished by their brown 
color. Haematoidin-crystals are sometimes found 
also. 

(h) Foreign substances. — Pus originates either 



THE EXAMINATION OF FLUIDS. 157 

from a free surface, as a mucous or serous membrane, 
an ulcer, etc., or from tlie midst of the tissue ; in 
either case it often has mixed with it elements from 
its place of origin. Since the latter is frequently 
entirely unknown, and is the object of search, it is 
evident that the foreign substances found in pus pos- 
sess at once great diagnostic value. 

The important question whether an abscess is 
connected with bone or not is often decided by a 
careful microscopical examination ; the irregular frag- 
ments of bone which are found in pus in these cases, 
and which often possess indented surfaces of absorp- 
tion — the so-called Howship's lacunae — then furnish 
absolutely certain proofs. They are very sharply de- 
fined through the marked glistening appearance of 
their calcified basement substance, and through their 
characteristic stellate, or spindle-shaped, bone-corpus- 
cles; they may often be found with a low power. 
If necessary the preparation is cleared up with a 
solution of potash, so that the pus-cells disappear ; if 
the pus is not too thick it should be allowed to set- 
tle, and the sediment should be examined. 

In other cases we may find bits of food, where- 
upon we infer the presence of a communication with 
the alimentary canal ; or epithelium, tumor-elements, 
etc., are encountered, so that we are often enlightened 
to a surprising extent in this way with regard to the 
diagnosis, while the information is of direct value 
from a therapeutical stand-point. As an example : In 
the pus contained in an abscess which developed be- 
side the thyroid gland of a woman, after confinement, 
there were found large numbers of flat epidermal 



158 THE USE OF THE MICROSCOPE. 

cells and a good deal of cliolesterin ; the diagnosis of 
suppurating bronchial cyst was at once made, and the 
sac was extirpated. Suppurating echinococcus-cysts, 
also, are frequently recognized correctly only by a 
microscopical examination of the pus, in which either 
entire scolices, the characteristic hooks, or the strati- 
fied, homogeneous membranes are demonstrated. 

(<?) ScJiizomycetes and Actinomycetes. — Micrococci 
are found in very large numbers in pus from acute 
abscesses,* being generally arranged in the form of 
chains, which are visible either when examined in the 
fresh state without further treatment or after they 
have been stained by the familiar processes. Micro- 
organisms do not constantly occur in chronic suppura- 
tions ; reference is, of course, made only to those cases 
in which the pus does not communicate with the 
external air, since saprophytic organisms are then 
always present. 

In tuberculous abscesses (periarticular and articu- 
lar abscesses, fungous arthritis, strumous and carious 
suppuration, cheesy and purulent lymphadenitis, etc.) 
tubercle - bacilli are naturally often observed, and 
they always possess a pathognomonic significance 
under such circumstances. 

On the other hand, they are not so invariably 
present as in pulmonary phthisis. Bacilli can not be 
found in many genuine tuberculous abscesses; ac- 
cording to Schlegtendal ("Fortschr. d. Med.," 1883, 
S. 537), they are met with in only about one-half of 
the cases. 

Less often other organisms appear in pus ; from 

* Comp. Ogston, "ttber Abscesse," " Arch, far clin. Clrir." Bd. 25. 



TEE EXAMINATION OF FLUIDS. 159 

these are excluded those which are purely accidental 
and develop subsequently — for example, the micro- 
cocci which cause the color of " blue pus." 

The actinomycetes — which were found in man, 
first by Langenbeck, and later by J. Israel, Ponfick, 
and others, and in cattle by Bollinger * — may be re- 
cognized microscopically in pus as gelatinous, miliary 
granules ; on squeezing the granules their peculiar 
structure at once becomes apparent, so that staining 
is unnecessary. 

4. The Ukeste. — In discussing the microscopical 
examination of the formed elements of the urine we 
consider first 

(a) Precipitates and Crystals. — The urinary salts, 
especially urate of sodium, which are normally held 
in solution at the temperature of the body, are pre- 
cipitated (if they are present in considerable quanti- 
ties) as soon as the urine becomes cool, in the form 
of fine, often rather irregular, granules. The author 
has frequently remarked that these are regarded as 
micrococci by novices, their quivering molecular 
movement being mistaken for vital locomotion. 

The salts of uric acid readily dissolve on warming 
slightly, also on the addition of acids, through the 
action of which uric acid separates in the form of 
characteristic crystals, generally prismatic in shape, 
and frequently of a brownish color. In febrile con- 
ditions, as in gout, urate of sodium is usually much 
increased ; the uric acid often separates spontaneous- 
ly in the urine a short time after it is voided, that is, 
when no acids have been added. This was formerly 

* Comp. Ponfick, "Die Actinoinycose," Berlin, 1882. 
8 



160 THE USE OF THE MICROSCOPE. 

described incorrectly as "acid fermentation of the 
urine." The process is simply this : The acid phos- 
phate of sodium, under the decomposing action of 
the urate, is changed into basic phosphate of so- 
dium, while the uric acid is set free. A genuine 
acid fermentation of the urine, with an increase in 
its acid reaction, occurs in diabetes (Voit and Hof- 
mann). 

Simultaneously with the appearance of the uric 
acid crystals there often occurs a separation of oxa- 
late of lime in the form of small, shining octahedra, 
which, when viewed from above, resemble envelopes. 
When the urine is abnormally rich in oxalates, we 
speak of " oxaluria." Oxalate of lime, as is well 
known, forms the principal component of an import- 
ant class of urinary calculi. In the urine after excre- 
tion from the body there occurs regularly an alkaline, 
or ammoniacal, fermentation, that is a decomposition 
of urea to form carbonate of ammonium, which change 
is effected through the agency of an amorphous fer- 
ment which was isolated by Musculus. This ferment, 
however, is itself always produced by micro-organ- 
isms, that is, by schizomycetes. If these organisms, 
or their germs, get into the urine that is contained 
within the bladder (this they do principally during 
catheterization) the alkaline fermentation may take 
place at once in the bladder, especially if the urine is 
retained, as in vesical paresis. During alkaline fer- 
mentation the urine becomes verv turbid. This tur- 

%/ 

bidity (aside from the presence of schizomycetes) is 
due to 

1. Phosphate of ammonium and magnesium (triple 



THE EXAMINATION OF FLUIDS. 161 

phosphates), in the well-known form of coffin-lid 
crystals, which are immediately soluble in acids. 

2. Urate of ammonium, in the shape of " morning- 
stars," or balls covered with fine projections. 

3. Phosphate of lime, an amorphous precipitate. 
In certain pathological conditions, beside the sub- 
stances already mentioned, which may be present 
in enormous quantities ("urinary gravel"), various 
other crystalline and granular precipitates are found, 
such as regular hexagonal tables of cystin (in cysti- 
nuria), xanthin and allied bodies, tyrosin in the shape 
of needles gathered into sheaves, and generally of a 
yellowish color (especially in acute yellow atropy), 
sulphate and carbonate of lime, etc. ; all these pre- 
cipitates can generally be very easily distinguished by 
means of simple micro-chemical reactions. Chemical 
text-books must be consulted for information upon 
this subject. 

(b) Urinary Casts* — Three principal varieties 
of casts are to be distinguished, hyaline, waxy, and 
brown. The hyaline consist of a substance which 
has a very delicate outline and is perfectly transpa- 
rent, so that they may be easily overlooked ; they 
are sometimes rendered more apparent by reason of 
adherent fat-granules. Their breadth frequently only 
equals the diameter of a red blood-corpuscle, but as 
a rule it exceeds it. They are found in albuminous 
urine during the most various pathological processes, 
even in cases in which neither inflammatory, nor other 

* Casts were discovered in the urine by Henle, and, as a result of 
examinations made on the cadaver, they were soon recognized as molds 
of the urinary tubules. " Zeitschr. fur rat. Med.," Bd. 1. 



162 THE USE OF THE MICROSCOPE. 

similar conditions are present in the kidneys, as in 
many febrile affections, in icterus, etc. They are 
accordingly to be regarded as the attendants of every 
case of albuminuria, though mild in character. The 
waxy casts, on the other hand, when found in con- 
siderable numbers, are of positive diagnostic import- 
ance ; they should always be regarded as sure signs 
of disease of the kidneys, and may accompany con- 
gestion as well as genuine nephritis. 

Waxy or colloid casts are composed of a substance 
which presents sharp outlines and is more or less 
glistening, or they may be slightly clouded by de- 
posits of very fine granules in their interior ; in the 
latter case they are distinguished as a special variety, 
receiving the name " granular " casts. 

They are of quite variable width, reaching five 
one-hundredths of a millimetre and more ; their 
shape is, as a rule, perfectly cylindrical, or cruciform 
on transverse section, while they have sometimes ir- 
regular, serrated borders, especially in acute nephritis. 
They are often filled with small round cells and 
fat-drops, and sometimes also with epithelial cells; 
there are forms of casts, in fact, which consist almost 
entirely of epithelial cells that are more or less firmly 
coherent (epithelial casts). The old expression " fibri- 
nous " cast has properly been given up by all ; the 
substance composing these casts is essentially different 
from fibrin, since they are neither redissolved by 
acetic acid, nor do they swell when placed in it, but 
they merely tend to lose their glistening character 
and dark contours, and finally their granular contents, 
through the action of the acid. They then appear as 



THE EXAMINATION OF FLUIDS. 163 

pale hyaline casts. Casts are stained a slight yellow 
color by the action of iodine, waxy casts frequently 
becoming even dark yellow or reddish brown. 

"VVe shall not enter here into the controversies re- 
garding the origin of casts ; the hyaline appear to 
be a direct product of exudation, while the waxy 
may, at least in part, come from disintegrated epithe- 
lial cells. 

A peculiar form of small, brownish casts was 
found in the urine by Eiegel * during the first few 
days after the occurrence of fractures ; he explains 
these, with great probability, as products of the fibrin- 
ferment which has got into the circulation. In these 
cases there are usually found also larger or smaller 
masses of fat, which collect in the form of small drops 
in the upper layers of the urine. This fat is intro- 
duced into the circulation through the agency of the 
wound, forms emboli in the renal vessels, and is then 
gradually excreted with the urine. In renal haem- 
orrhages and hemorrhagic nephritis there are often 
observed brown casts, which are stained with blood 
coloring-matter, and frequently also real blood-casts. 

(c) Pus- and Mucus-cells. Epithelial Cells. — Lym- 
phoid cells, pus- and mucus-corpuscles often appear in 
the urine ; they may come from the kidneys or urinary 
passages, or they may originate in an abscess which 
has ruptured somewhere into the urinary tract. Epi- 
thelial cells also are often found, the source of which 
can not always be positively determined. In urethral 
and vesical catarrh, epithelial cells are sometimes 

* u tlber das Verhalten des Urins bei Knochenbruchen." " Deutsch. 
Zeitschr. fur Chir.," Bd. 10. 



164 THE USE OF THE MICROSCOPE. 

found, within which one or more lymphoid cells are 
inclosed ; it was formerly supposed that these arose 
in an endogenous manner, but they are now regarded 
as having been invaginated, that is, they have pene- 
trated subsequently into the interior of the epithelial 
cells (Volkmann, Steudener). Cells containing fat- 
granules appear but seldom in the urine; Leyden 
found them in acute nephritis. 

(d) Tumor-elements are not difficult to recognize 
if some care be used, but this only applies to the ob- 
server who has become thoroughly acquainted with 
the epithelial elements of the kidney and urinary 
passages. The extremely multiform epithelial cells 
of the bladder (sometimes of very large size), which 
are provided with several nuclei, have often been 
mistaken for cancer-cells. 

Diphtheritic and tuberculo-caseous masses appear 
in the urine; they generally come from the blad- 
der. 

(e) Entozoa occur with extreme rarity in the 
urine ; these are echinococci, as well as filaria (the 
latter have hitherto been found only in the tropics in 
cases of chyluria), also the ova of Distoma hcemato- 
bium. Many errors in observation have occurred in 
this field ; a certain author, for example, described 
the ova of Strongylus gigas, which he professed to 
have seen in the urine, but on closer examination it 
turned out that he had mistaken for ova the granules 
of Semen lycopodii, which had got into the specimen 
through want of cleanliness. 

(/) Vegetable Parasites. — Sarcinae are found in 
the urine only in very exceptional cases. 



THE EXAMINATION OF FLUIDS. 165 

In order to prove the presence of bacteria and 
micrococci in the urine, it is of course necessary to 
examine a perfectly fresh specimen ; normal urine 
is always free from such organisms, but within a 
few hours after it has been voided they are found 
in great numbers. They are observed in the largest 
quantities, as was first proved by Traube, in fresh- 
ly passed urine, in those cases in which alkaline de- 
composition has occurred in the bladder after cathe- 
terization, and in consequence cystitis has developed. 
There are then found multitudes of triple phosphate 
crystals, lymphoid cells, and immense swarms of rods 
and granules which stain deeply with basic aniline 
dyes, and are often rolled together in large zoogloea- 
masses. In these cases the schizomycetes, the germs 
of which have entered the bladder by means of the 
catheter, very often wander through the ureters into 
the renal pelvis and true parenchyma of the kidney, 
and are then found in the interior of pyelo-nephritic 
abscesses (Klebs). In different infectious diseases, 
especially in metastatic abscesses, the organisms make 
their way from the blood into the urine, as can be 
demonstrated with certainty; as yet, however, few 
reliable clinical investigations on this subject have 
been reported. 

Those forms of schizomycetes which furnish the 
ferment that decomposes urea, never, as far as is now 
known, pass from the blood into the renal excretion 
(probably because they do not exist in the blood), 
but are always introduced from without. On the 
other hand, they must be present in the alimentary 
canal, for the urea which has entered the intestine 



166 THE USE OF THE MICROSCOPE. 

(in cases of uraemia) is rapidly transformed into car- 
bonate of ammonium. 

The gonococci, which occur in the urine in gonor- 
rhoeal cystitis, do not decompose the urea. 

I have several times demonstrated the presence 
of tubercle-bacilli in the urine in tuberculosis of the 
kidneys and urinary tract, though at first only in the 
cadaver; later they were frequently observed and 
utilized for purposes of diagnosis. The prognosis of 
tuberculous affections of the urinary organs is gener- 
ally a very unfavorable one. 

5. Seceetioists of the Genital Teact. (a) Vagi- 
nal Secretion. — The secretion of the vagina is a fluid 
which contains more or less abundant large, partially 
corneous, epithelial cells, and also some round cells. 
The latter vary in dimensions from the size of white 
blood-corpuscles up to forms which are four or five 
times larger ; the larger round cells generally contain 
numbers of fat-granules. 

A great many micro-organisms appear in the vagi- 
nal secretion ; all the conditions for the rich develop- 
ment of both harmless and injurious parasites are pres- 
ent here, just as in the mouth. Among the harmless 
ones we must always include the Trichomonas vagi- 
nalis, discovered by Donne in the vaginal mucus, an 
infusorium provided with flagella and cilia and capa- 
ble of active motion. Mold-fungi also develop upon 
the vaginal mucous membrane, especially in pregnant 
women ; if they form in larger masses they cause 
white patches and a slight inflammation of the 
membrane. This, according to Haussmann, is the 
Oidium albicans, or thrush-fungus, through infection 



TEE EXAMINATION OF FLUIDS. 167 

with which the thrush of new-born infants may be 
produced. Many of the schizomycetes that appear 
in the vaginal secretion have hitherto not been dis- 
tinguished from one another at all, or only in an im- 
perfect way. With reference to the micrococci of 
gonorrhoea, see below under cj these, too, have not 
yet been so exactly described that they can aid the 
diagnosis in doubtful cases. 

(5) Fluids from the Uterus. Dysmenorrhea! 
Membranes. Decidual Remains. The Diagnosis of 
Carcinoma of the Uterus. — Beside the normal mucous 
plug of the cervix uteri, which contains only a few 
lymphoid cells, there is found during inflammatory 
conditions a fluid secretion of the uterine mucous 
membrane, often purulent, in which, in addition to 
lymphoid cells, many cylindrical epithelia appear, 
generally without cilia. 

The menstrual discharge consists mostly of blood ; 
the lochia also contain numerous elements from the 
placental remains, especially from the deepest layer of 
decidua, which are retained in the uterus after deliv- 
ery. The epithelial cells from the lower ends of the 
glands, with their bright, almost vacuolated nuclei, 
are especially characteristic. 

If portions of the placenta have been retained we 
frequently find in the lochia smaller or larger pieces 
of it; the structure and form of the dendritic, 
branched placental villi (chorionic villi) are so char- 
acteristic, that they are always recognized as such at 
once. Deposits of lime are frequently observed in 
such placental remains, so that occasionally even firm 
concretions are expelled from the uterine cavity ; a 



168 THE USE OF TEE MICROSCOPE. 

histological examination of these at once furnishes a 
clew to their origin. 

In certain forms of dysmenorrhea, as is well 
known, membranes are cast off, while at the same 
time the patient often has pains resembling those of 
labor. Examination of these dysmenorrhoeal mem- 
branes invariably shows that they consist of actual 
pieces of the uterine mucous membrane ; even the sac- 
ciform glands and their openings upon the lining sur- 
face of the uterus can be demonstrated. Such mem- 
branes were formerly called "menstrual decidua," 
and the question was also raised whether in these 
cases there was not really an early abortion. The 
histological examination decides if this is the fact 
or not. For the minute structure of the decidua 
of pregnancy, that is of the uterine mucous mem- 
brane as changed by conception, is clearly different 
and is perfectly characteristic of the condition. The 
interglandular tissue of the mucous membrane of the 
uterus always consists, as far as is at present known, 
of small round cells as large as lymphoid cells, con- 
taining very little protoplasm, and this is the case 
under all circumstances, as in the swollen state of 
menstruation, in dysmenorrhoeal membranes, in the 
different varieties of endometritis, in the swelling due 
to uterine myomata, etc. Pregnancy alone causes a 
characteristic change in the cells ; even at the begin- 
ning of this condition we find in the swollen mucous 
membrane the familiar large decidual cells, from five 
to ten times the size of leucocytes, rich in protoplasm, 
round or polygonal in form, and provided with pro- 
cesses. The cells of the decidua retain this size and 



THE EXAMINATION OF FLUIDS. 169 

shape until the end of pregnancy. The decidual tis- 
sue, if the glands be excepted, resembles many forms 
of large-celled sarcoma. In extra-uterine foetation, also, 
a swelling of the uterine mucous membrane regularly 
occurs, and fragments of the same are often expelled ; 
the characteristic formation of the decidua (its large 
cells) can always be demonstrated in these cases.* 

Carcinoma, Erosion, or Adenoma? — In carci- 
noma of the uterus there are frequently found, sus- 
pended in the fluid which has exuded from the can- 
cerous ulcer, cellular elements, or even pretty large 
fragments and shreds, the structure of which as 
viewed through the microscope assists in establishing 
the diagnosis. In doubtful cases, however, where the 
question is whether we have to do with carcinoma 
or with a simple erosion, the examination of the se- 
cretion alone will never be sufficient; in such in- 
stances small bits are frequently excised, by the his- 
tological study of which the diagnosis should be 
confirmed. We shall introduce here a few observa- 
tions upon this subject because of its practical im- 
portance. 

The floor of an erosion consists of granulation- 
tissue, which is usually covered by several layers of 
epithelial cells; gland-like prolongations provided 
with lumina extend from this epithelium into the 
depths of the granulation-tissue; these may, how- 
ever, be solid epithelial columns, which subdivide 
and unite with one another to form an irregular net- 
work. 

As is at once evident, this structure closely re- 

* Wyder, " Arch, fur Gynakologie," Bd. 13. 



170 THE USE OF THE MICROSCOPE. 

sembles cancer, although it occurs in perfectly sim- 
ple benign erosions. A careless observer who, with- 
out further evidence, makes the diagnosis of cancer, 
may readily occasion in this way a great deal of un- 
happiness; he will undertake mutilating and dan- 
gerous operations in cases in which a radical extirpa- 
tion is not indicated. Another element must be 
present before we are justified in making the momen- 
tous diagnosis of cancer. The secondary epithelial 
proliferation, which extends into the granulation-tis- 
sue and is often perfectly atypical, is not limited to 
erosions of the uterus, but appears very often and in 
the most dissimilar localities, as in the skin, liver, 
lungs, etc. ; it may occur at any spot where granula- 
tion-tissue comes into direct contact with epithelial 
surfaces. The atypical epithelial outgrowth does not 
vary at all with the character of the original affection 
which has led to the formation of the granulation- 
tissue ; the process is a perfectly benign and harmless 
one, and would be of but little interest to us from 
a practical stand-point, if the structure of cancer, es- 
pecially at the beginning of its course, did not bear 
the most perfect resemblance to the innocent atypical 
growth of epithelial cells. 

Cancer has even been defined, from a histological 
point of view, as " an atypical proliferation of epithe- 
lium" (Waldeyer). This definition, however, is not 
sufficient ; we must add to it, as being necessarily the 
most important peculiarity of cancer, the words " of a 
malignant character." With this expression we leave 
the purely histological field, for we can not recognize 
directly the " malignant character," either in the cell 



THE EXAMINATION OF FLUIDS. 171 

or in the tissue. Nevertheless the microscopical ex- 
amination again proves the fact of malignancy ; for 
the malignancy of the process is shown if it forces its 
destructive toay through various tissues without hin- 
drance, while an innocent growth remains limited to 
the tissue from which it originated, and either leaves 
the neighboring parts entirely intact, or merely pushes 
them aside. If we discover in the uterus, for ex- 
ample, that the process is not confined to the mucous 
membrane, but that it also invades the muscular 
tissue, and that the muscle is partly replaced by gran- 
ulation-tissue,* traversed by processes of atypical 
epithelium, we have before us a clearly malignant 
element, and then only do we make the positive 
diagnosis of cancer. 

Fragments removed for the purpose of histological 
examination must also include at least a part of the 
muscular layer ; the diagnosis of cancer can not be 
surely established unless it has been shown that the 
muscle is affected. In this respect I am opposed to 
C. Ruge, f who proposes a very simple method of dis- 
tinguishing a cancerous ulceration from an innocent 
erosion. 

Ruge affirms that in cancer the epithelial columns 
are solid, while in simple erosions they contain lumina, 
and this is certainly correct in many instances. But 
if he means that this difference is present without ex- 
ception, and that use can therefore be made of it for 
purposes of diagnosis, he is entirely in error ; for, on 

* The stroma in young cancerous growths is usually composed of 
granulation-tissue. 

tC Ruge, " Berlin klin. Wochenschrift," 1878. 



172 THE USE OF THE MICROSCOPE. 

the one hand, in many cases of genuine malignant 
cancer the cell-columns possess most beautiful and 
regular lumina, and, on the other, solid epithelial 
plugs are very often observed in innocent erosions, as 
may be readily shown by examinations of bodies. 
The criterion proposed by Ruge must accordingly 
be regarded as entirely unreliable for the diagnosis 
of cancer. 

These maxims with regard to the decision of this 
question should be observed in the case of all the 
other organs ; as long as we find atypical epithelial 
growths in localities in which epithelium has always 
existed, as in the skin, mucous membranes, in all true 
glands, etc., we must always show positive proof of 
malignancy before we make the diagnosis of cancer. 
If, however, these atypical epithelial processes occur 
elsewhere — in muscle, for example, in bone, or in the 
lymph-glands — then the eroding, or metastatic, charac- 
ter of the affection has been demonstrated at once eo 
ipse, and the diagnosis of cancer is necessarily made. 

Many authors use the term epithelioma as synony- 
mous with cancer, which, according to my opinion, is 
not to be commended. The word " epithelioma " we 
should retain as the general designation for epithelial 
tumors of all Muds / we have the word " cancer " for 
such of them as are malignant, while the expression 
" adenoma " is best applied only to benign growths* 

The cause of the malignancy of cancer is not yet 
known. Nevertheless, as the results of thousands of 

* Epithelial elements are found in teratoid tumors, even in the midst 
of other tissues, without the growths being considered as malignant. 
In myomata of the uterus cysts have rarely been discovered which were 
lined by ciliated epithelium. 



THE EXAMINATION OF FLUIDS. 173 

observations, we can at once, and with great certainty, 
give a fatal prognosis in all cases in which it was 
necessary to make the diagnosis of cancer according 
to the principles before stated ; if total extirpation 
is not performed the patient will soon die. 

To this practical rule there are only very rare ex- 
ceptions ; there are certain forms of superficial cancer 
of the skin (rodent ulcer) which may run an extremely 
protracted course. 

On the other hand, we can deny the presence of 
immediate danger if the tests of malignancy already 
detailed are absent ; it is, of course, not impossible for 
a cancer to develop subsequently from a growth that 
was originally innocent, but positive danger is only 
imminent when the malignant character of the pro- 
cess has been demonstrated. 

The diagnosis may, under certain circumstances, 
become difficult, as when an atypical epithelial growth 
is combined with a syphilitic or tuberculous ulcera- 
tion ; special caution is advisable in such cases, and it 
is better to first await the result of vigorous local 
treatment. 

(c) Gonorrhoea! Secretion. — Neisser* found a spe- 
cific variety of micrococcus in gonorrhoeal pus, which 
is distinguished by its forming small groups, in which 
the single granules are relatively far apart ; they fre- 
quently lie upon the exterior and in the protoplasm 
of pus-corpuscles. The same micrococci are found in 
the secretion of specific conjunctivitis. They are 
readily stained in the usual manner with aniline dyes. 
The characteristic points about the gonococci are not 

* STeisser, " Med. Centralbl.," 1879. 



174 THE USE OF THE MICROSCOPE. 

yet sufficiently clear, so that we are not in a position 
to distinguish with certainity the micrococci of gon- 
orrhoea when they are mingled with other micro- 
organisms, as in the vaginal secretions. 

(<#) Semen and Prostatic Secretion. — The highly 
characteristic, active spermatozoa are found in the 
semen ; they may still be demonstrated very well, as 
a rule, in dried semen (semen-stains) by moistening 
it with salt-solution. If we find in doubtful cases 
small glistening bodies which look like the heads of 
spermatozoa, and also fine threads resembling tails, 
but no perfect spermatozoa, we must not be led into 
the error of drawing a positive conclusion from the 
fragments discovered. Similar small corpuscles, or 
threads, are easily shown in dry spots of any character 
whatsoever, and they may have every possible source ; 
the nature of the semen-stain is only positively rec- 
ognized when perfect seminal bodies, with heads and 
tails in continuo, can be demonstrated. If sperma- 
tozoa are not found in the semen, care should be taken 
to distinguish the temporary from the permanent form 
of azoospermia (sterilitas masculind) ; if several ejacu- 
lations are provoked, one soon after another, the fluid, 
according to the testimony of a number of observers, 
is finally entirely devoid of spermatozoa. The secre- 
tion of the testicles is then temporarily exhausted 
and the fluid ejaculated comes only from the seminal 
vesicles and the prostate. 

The prostatic secretion is generally mingled with 
the semen, but it is sometimes discharged independ- 
ently by pressure upon the prostate, as during defe- 
cation. It often contains, beside the laminated amy- 



THE EXAMINATION OF FLUIDS. 175 

loid corpuscles, a large number of octahedral or lan- 
cet-shaped crystals, which, like the asthma-crystals in 
bronchial secretions, represent the phosphatic salt of 
an organic base — the so-called Schreiner's base. They 
have long been known in the semen as spermatic 
crystals ; Fiirbringer * has shown that they originate 
in the mixed prostatic secretion and that they are the 
source of the peculiar odor of the semen. They may 
often be demonstrated in freshly discharged seminal 
or prostatic secretion, but otherwise only after it had 
stood for some time; they always form in large 
quantities if ammonium phosphate is added to the 
seminal or prostatic fluid. 

6. Contents of the Stomach akd Intestine. — 
The microscopical examination of vomited matters 
and of the stools has long been practiced, and in many 
cases it furnishes important diagnostic aids. 

(a) Remains of Food are, of course, always 
found in large quantities. During a meat diet striated 
muscular fibers occur regularly in the dejections 
(Frerichs) ; likewise the cellulose membranes are 
found during a vegetable diet, while, under the nor- 
mal conditions of digestion, starch is either not pres- 
ent in the faeces, or only in very small amount. 
Most of the starch-granules are at once perfectly ex- 
tracted in the stomach; they then no longer stain 
blue with iodine, but take only a light-yellow color. 
Whenever a considerable quantity of starch is found 
in the evacuations of the intestine, a pathological con- 
dition is present, generally attended with diarrhoea. 

Among the animal food-elements tendons, apo- 

* Furbringer, " Zeitschr. fur klin. Med./' Bd. 3. 



176 THE USE OF THE MICROSCOPE. 

neuroses, fragments of large arteries, etc., long resist 
the digestive forces ; large quantities of such mate- 
rials are often found, not only in the dejections of in- 
sane, or especially gluttonous individuals, but in the 
stools of men who are quite prudent in their eating. 
These masses, occurring in the form of so-called " in- 
testinal concretions," not infrequently occasion seri- 
ous anxiety both to the patient and to the physician ; 
but on microscopical examination it is at once recog- 
nized that this is unfounded. Undigested vegetable 
matters are often voided ; Virchow has called atten- 
tion to the frequent appearance of orange-pulp in the 
stools. 

(b) Epithelial Cells, Mucus, etc. — The epithelial 
and gland-cells of the alimentary canal are frequently 
met with in the contents of the stomach and intestine 
(but generally much altered), without this discovery 
possessing any particular significance. Large masses 
of mucus, which are apt to contain leucocytes, mucus- 
corpuscles, etc., denote the presence of a gastric or 
intestinal catarrh. 

Sometimes, in addition to the mucus, collections of 
fibrin appear in the stools, either in the form of mem- 
branes or of dendritic, branched processes that unite 
in the shape of a net-work. These are to be regarded 
as the products of a pseudo-membranous inflammation 
of the colon which, as is well known, sometimes as- 
sume the appearance of a net- work. The evacuation 
of these masses, which may attain considerable dimen- 
sions, is sometimes attended with severe pains like 
those of labor. They usually contain fibrin, as well 
as mucus, and therefore only partially dissolve in 



THE EXAMINATION- OF FLUIDS. 177 

acetic acid ; nothing else is found in them except round 
cells, or their debris. 

(c) Entozoa of different kinds appear in the ali- 
mentary canal, many of them being unimportant para- 
sites, while others are highly dangerous. The animals 
themselves, or portions of the same, are found in the 
stools, but their ova are valuable as a means of recog- 
nizing them in the dejections ; accurate descriptions 
and illustrations of these will be found in text-books 
upon pathological anatomy, also in the well-known 
works of Leukart, Braune, and Davaine. 

In the year 1880 Perroncito made the important 
discovery that the " tunnel-disease," which decimated 
the St. Grothard workmen, was caused by the Anchylo- 
mum duodenale (or Anguilhda intestinalis and Pseii- 
dorliabditis stercoralis) ; the eggs of these parasites 
are found in immense numbers in the stools of the 
patients, and are perfectly characteristic. In the so- 
called " mountaineers' anaemia " also, which prevails 
in the mines of Hungary, he found the same parasites. 
Perhaps a careful examination of the stools in other 
hitherto problematical diseases will furnish new light. 

(d) Vegetable Parasites. — Sarcinae have long been 
recognized in the contents of the stomach, especially 
in cases of dilatation; no clinical importance is at- 
tached to them. Of the other vegetable parasites, 
micrococci, bacilli, etc., appear in the stomach only in 
small numbers, but very large masses of yeast fungi 
are often seen. 

Yeast-fungi are likewise present in the contents of 
the intestines and in the dejections, but micrococci and 
bacilli, both large and small, are found in extremely 



178 THE USE OF THE MICROSCOPE. 

large numbers ; certain forms of rods have been de- 
scribed by Ferd. Colin as Bacillus subtilis. Among 
these confused masses of micro-organisms it has thus 
far been impossible to distinguish any specific patho- 
genic forms, with the exception of the tubercle-bacilli 
characterized by their peculiar relation toward stain- 
ing processes. 

There are several forms of organisms in the con- 
tents of the intestine which turn blue on the addition 
of iodine ; of special interest is the Clostridium butyri- 
cum (Prazmowsky *), large quantities of which are 
found in the lower section of the ileum and in the 
colon (but not in the upper portions of the alimentary 
canal), especiallly after a vegetable diet (Nothnagel f ). 
Their size is variable, something like that of yeast- 
fungi, though their form is different; they appear 
either in the shape of rods, often drawn out into sin- 
gle or double-pointed extremities, or of elliptical, spin- 
dle-, or lemon-shaped bodies. They are frequently 
collected together into chains or groups. They are 
stained blue with iodine, either entirely, or only at 
their centers, while their extremities (or entire per- 
iphery) take a yellowish tinge. They are probably 
identical with the Bacillus amylobacter of Van 
Tieghem, and represent the ferment of the butyric- 
acid fermentation which occurs in the contents of the 
intestine. Nothnagel has also described still smaller 
forms, which occur in the dejections and may be 
stained with iodine. Tubercle-bacilli are very often 

* Prazmowsky, " Untersncmmgen iiber Entwickelungsgeschichte 
und Form ent wick elung einiger Bacterien," Leipzig, 1880. 
t Nothnagel, " Zeitschr. fur klin. Med.," Bd. 3. 



THE EXAMINATION' OF FLUIDS. 179 

encountered in cases of tuberculous ulcers of the in- 
testines, in the contents of the gut, and of course in 
the faeces. Numbers of tubercle-bacilli are regularly 
present on the floors of these ulcerations ; they then 
become mingled with the intestinal contents, and may 
be used for making a diagnosis during the examina- 
tion of the feces.* It should be remembered that 
the sputum of phthisical patients may be swallowed 
and may also give rise to a mixture of tubercle-bacilli 
with the faeces ; in every case a tuberculous affection 
of the body is certainly established by the finding 
of these organisms. The other bacilli which appear 
in the contents of the intestine are at once decolorized 
by treatment with acids ; but certain forms of large, 
round micrococci are observed in the faeces, which, 
like tubercle-bacilli, retain their color for a long time 
when treated with acids. These bodies are regarded 
by Koch as spores which possess exceptional capaci- 
ties for staining, since it is well known that the spores 
of most bacilli with which we are acquainted do not 
stain. By reason of their globular shape they can 
certainly never give rise to diagnostic errors. 

7. Exudations. The Contents of Cysts. — The 
microscopical examination for diagnostic purposes of 
exuded fluids, cyst-contents, and the like, is very often 
undertaken at the present time, on account of the 
great frequency with which exploratory puncture is 
resorted to. 

The technique of these examinations is as simple 
as the difficulty of estimating properly the diagnostic 
value of the discoveries is great ; it is usually neces- 

* Lichtheim und Giacomi, " Fortschr. der Med.," 1883, S. 3 and 150. 



180 THE USE OF TEE MICROSCOPE. 

sary merely to examine the sediment according to 
the methods already described, or to stain the dried 
preparation. 

Large quantities of fat occur not infrequently in 
exudations, and cause them to assume an opalescent 
or milky character. The fat is sometimes contained 
in the fluid, as in chyle, in the form of minute, irreg- 
ular, slightly glittering granules, intimately combined 
with albuminous bodies, so that it is not recog- 
nized as such without further tests ; it is only after 
adding acetic acid or alkalies that the albuminates are 
dissolved and the fat runs together to form large, 
shining drops. This so-called hydrops cliylosus is 
never seen except when chyle is poured out into 
the abdominal or thoracic cavity, or as the result of a 
wound, or obstruction to the flow of the chyle in the 
lacteals or thoracic duet.* In other cases the fat comes 
from disintegrated fatty cells, and is at once recog- 
nized by its small, glistening granules (liydrops adi- 
posus) ; this is observed in chronic inflammation, as 
well as in carcinoma of the serous membranes. 

Serous effusions also occur, which only show an 
opalescent or milky cloud, due to the presence of al- 
buminous granules. Red blood-corpuscles are found 
in variable quantities, and often in an altered con- 
dition, as decolorized, shrunken, etc. 

Lymphoid cells are almost never absent, only they 
are far less numerous in simple transudations than in 
inflammatory exudations. Active amoeboid move- 
ments may be observed in them, but in many cases 

* Compare Quincke, " Ueber fetthaltige Transsudate," " Deutsch. 
Arch, fur klin. Med,," Bd. 16, 



THE EXAMINATION- OF FLUIDS, 181 

they are already dead ; they are frequently filled with 
numerous fat-granules. 

Endothelial cells appear in serous effusions partly 
single, partly in coherent plates, often also filled with 
fat or transformed into globular bodies ; they are pro- 
vided with one or more nuclei, and sometimes contain 
vacuoles also. 

Epithelial cells, cylindrical, polygonal, or flat, are 
observed in the contents of cysts ; they often furnish 
clews to the diagnosis of the origin of cysts. Ciliated 
epithelial cells are sometimes found, especially in 
monolocular cysts. 

Tumor-elements, which are mingled with exuda- 
tions, are generally quickly precipitated, and this hap- 
pens even within the body, so that if punctures be 
made high up the cells may be missed entirely, while 
in the more dependent parts they are present in 
abundance. 

This applies chiefly to cancer, the cells of which 
are distinguished by their variable size, their large 
nuclei, and their polymorphous forms; vacuoles are 
also seen very frequently. However, it is not usually 
advisable to make a diagnosis of cancer from finding 
a few cells ; it is much better for the beginner to 
leave the diagnosis in doubt until the cells are found 
collected together into clusters or balls. Quincke* 
affirms that the glycogen-reaction can probably be 
employed in forming a diagnosis ; this reaction often 
occurs in cancer-cells, while endothelial cells usually 
appear to be devoid of glycogen. 

* Quincke, "Ueber Ascites," "Deutsch. Archiv. fur klin. Med.," 
Bd. 30. 



182 THE USE OF THE MICROSCOPE. 

He who desires to establish such diagnoses must 
first examine thoroughly the formed elements of the 
different varieties of serous effusions as they appear 
in the cadaver, since otherwise errors are certain to 
occur. The variations of form shown by the endothe- 
lial cells and their derivatives in ordinary subacute 
or chronic inflammations of serous membranes are 
often very surprising to the novice, and may readily 
give rise to a false diagnosis of cancer. 

Schizomycetes have as yet been seldom examined 
in effusions ; possibly a more careful study of these 
will furnish other important aids to diagnosis. 

In the pleurisy and pericarditis accompanying 
acute pneumonia, quantities of pneumococci frequent- 
ly develop, which, in doubtful cases, may very ma- 
terially assist in the diagnosis. By puncturing the 
lung-substance Gunther and Leyden succeeded, in 
two cases of acute pneumonia, in finding micrococci 
in the alveolar exudation of the living subject; 
Gunther notes especially in his case the presence 
of " colorless capsules " around the pneumococci. 
Of the animal parasites, the echinococcus is of special 
importance in this connection; the chitinous hooks 
and the stratified membranes are to be used as posi- 
tive microscopical proofs. In inspecting the sediment 
carefully we frequently find, even on macroscopic ex- 
amination, the separate or grouped scolices, appearing 
as white specks. 



VIL 

THE EXAMINATION OF SOLID ELEMENTS OF THE 
BODY, EXTIRPATED TUMORS, ETC. 

The microscopical examination of solid elements 
of tlie body, tumors, and the like, is effected accord- 
ing to the methods before described. The isolated 
elements are obtained either by examining the fluid, 
by scraping the cut surface, or by teasing, after pre- 
vious maceration. For this purpose always make, 
with a perfectly clean knife, a freshly cut surface / 
otherwise you run the risk of being constantly em- 
barrassed by numbers of accidental impurities that 
have collected on the surface. 

Sections are also prepared from the fresh speci- 
men by means of the curved scissors, razor, double 
knife, or freezing-microtome, and are examined at 
once in salt-solution ; Bismarck-brown and solution of 
iodine are mostly used for staining fresh sections. 

Specimens should always be examined first in a 
fresh condition, since this offers many advantages. 
Substances then show their natural transparency, and 
in this way we are best able to compare the histo- 
logical structures which we have found with the dif- 
ferences observed macroscopically. If there is any 
fatty degeneration present it is only seen in its full 
extent in the fresh preparation. 



184 THE USE OF THE MICROSCOPE. 

The freezing-microtome should be used in all 
cases in which it is desirable to prepare for demon- 
stration neat specimens that are suitable for stain- 
ing. It is generally possible in this way to obtain in 
a very short time entirely satisfactory, perfect sections 
which can be examined directly in salt-solution ; the 
nuclei are then stained very beautifully with Bis- 
marck-brown, the specimen is transferred from Bis- 
marck-brown to alcohol for a short time, and is then 
mounted in glycerin, or it is placed first in oil of 
cloves, and afterwards in Canada balsam. In this 
way, where haste is necessary, specimens affording a 
perfect view of the structure may be prepared within 
a few minutes, from organs freshly removed from the 
cadaver, as well as from extirpated portions of the 
living body, or even during an operation ; this, under 
some circumstances, is a very important advantage. 

In many instances, however, we are not successful 
in the examination of the fresh preparation; large 
and very delicate sections of fresh organs are apt to 
curl up, and are so extremely soft and easily destroyed 
that sometimes, in spite of the greatest care and much 
loss of time, it is not possible to transfer them intact 
to the slide and to spread them out well. Then the 
true value of hardening appears. Hardening (aside 
from special cases) is always effected by means of 
alcohol ; only, in the case of the nervous system and 
the eye, it is sometimes necessary to give up this 
hardening fluid, the effects of which are simple and 
easily controlled, and to make use of chromic acid 
and its salts (Muller's fluid). 

Hardening in alcohol is best accomplished by 



EXAMINATION OF TUMORS, ETC. 185 

placing small pieces of the substance in large quanti- 
ties of absolute alcohol ; in this way the concentrated 
spirit penetrates rapidly into the interior of the pieces, 
and, by immediately coagulating the albumins, effects 
a speedy fixation of the tissue-elements (compare 
page 24). 

The hardened specimens are then always cut with 
the microtome (compare page 13) ; a large number of 
successive sections are thus obtained rapidly, which 
can be treated in any desired manner. Always 
examine first in simple water and glycerin, and after 
that study the effect of reagents and staining. In 
many cases a single brief examination is sufficient, 
since we are frequently required only to classify, 
or to name, a given condition ; in this case it is suffi- 
cient to examine one preparation unstained, and an- 
other in which the nuclei have received any desired 
color. In other instances, however, we meet with 
unsuspected, often surprisingly new, structures and 
combinations; it is then advisable to store away a 
great many sections, preserving them in alcohol. 
New views, or new questions, often come up after the 
lapse of some time, which can be answered by some 
peculiar method of treatment such as had not pre- 
viously been thought of. 

As regards the method of procedure in isolated 
cases, and the value of the observations for diagnostic 
and pathological purposes, we should exceed the 
bounds of our little book if we undertook to treat 
this subject specially. This requires, besides clini- 
cal knowledge, an accurate training in pathological 
anatomy and histology, and great circumspection, 



186 THE USE OF THE MICROSCOPE. 

which can only be acquired through a thorough ex- 
perience with the subject. It may be laid down 
as a fundamental principle that a discovery should 
never be regarded as pathological in its character 
unless the specimen has in every case been directly 
compared with the normal organ, which has been 
similarly treated. The disregard of this maxim, 
which really sounds as if it were self-evident, has 
already introduced into science many gross errors, 
and leads practically to the most serious miscon- 
ceptions. Many new facts in normal histology 
have been discovered in the course of pathological 
examinations ; the endeavor was made to prove that 
the new objects, which were first represented to be 
pathological, were the cause or products of a certain 
disease, until it was ascertained later that these were 
perfectly normal structures that had been previously 
overlooked. This was the case, for instance, with the 
cells filled with fat-granules, which are found in the 
central nervous system of old fetuses and new-born 
infants ; these cells were first regarded as evidences 
of encephalitis, until it was afterward proved by 
Jastrowitz and Flechsig that they are necessary 
transitional forms in the normal development of the 
white substance. 

In pathological discoveries we are very often con- 
cerned with quantitative deviations from the normal / 
for example, with the proliferation of nuclei, clouding 
of the protoplasm (although this shows a certain 
amount of clouding even when normal), atrophy, and 
diminution in the size of cells. It is evident that in 
order to establish such gradual differences, a direct 



EXAMINATION OF TUMORS, ETC. 187 

comparison with the normal organ, which has been 
treated in precisely the same way, is positively neces- 
sary. Furthermore, objects are frequently found 
which are not exactly normal and yet which possess 
scarcely any pathological value — subnormal conditions. 
To this class belong especially the numerous senile 
degenerative changes, which are almost insignificant 
as long as they are not present in excess. They 
should always be observed, without, however, too 
great importance being assigned to them. 

The extent of the abnormal process sometimes has 
a very important influence upon the decision as to its 
pathological and clinical importance. The beginner 
is frequently inclined to estimate too highly the sig- 
nificance of his discoveries, and to regard as a very- 
extensive change one that covers the entire field of 
view of the microscope. Only repeated examinations 
can guard the novice from drawing erroneous con- 
clusions in this respect ; we gradually learn to judge 
correctly concerning the extent to which a process 
has spread over an organ by examining different por- 
tions of the organ, and especially by a systematic use of 
low powers. If, for instance, several shrunken glom- 
eruli are discovered in a kidney, do not decide at 
once, without further evidence, that the glomeruli are 
all shrunken, but first determine what per cent, of the 
glomeruli are altered, and how many still remain nor- 
mal, and whether the affection is a diffuse one, evenly 
distributed throughout the entire organ, or is localized; 
or whether the foci are more or less numerous and the 
intervening parts have remained quite normal, or also 
appear to be somewhat changed. If only a small por- 



188 THE USE OF TEE MICROSCOPE. 

tion of the organ Las undergone change, the process, 
although quite intense at the single spot in question, 
may be of very trifling clinical significance ; it should 
be remembered that even the sudden extirpation of 
an entire kidney from an otherwise healthy organism 
is tolerated without any marked disturbance. 

On the other hand a change that is really much 
less striking may, if it is diffused over the entire 
organ, be attended by very injurious consequences to 
its functions, and consequently to the whole body. 
This is the case, for example, in glomerulo-nephritis, 
a disease which is characterized by the proliferation 
of nuclei in the loops of the glomeruli, and by con- 
sequent imperviousness of the same ; in this way the 
flow of blood through the kidneys is greatly im- 
paired or is even reduced to a minimum. The inex- 
perienced observer may very easily overlook this 
important change completely, while the expert will 
have his attention at once directed to it through 
the contrast which the empty, and therefore large, 
glomeruli offer to the capillaries of the cortex and 
medulla when the latter are filled with blood. 

As regards the origin of certain processes, we 
should always bear in mind the difficulties and com- 
plications that are present ; we observe only that 
which has occurred, not that which is still in progress, 
and, without further evidence, a direct inference should 
not be drawn from the former concerning the latter. 

Since the discovery of the power of locomotion 
of cells, the emigration of white blood-corpuscles, etc., 
observers have been accustomed to be as reserved as 
possible in this respect, so much the more as a direct 



EXAMINATION OF TUMORS, ETC. 189 

practical interest does not usually attach to these 
questions. Some twenty years ago it was believed 
that we were much farther advanced in this matter 
than we are to-day ; then even the beginner was al- 
ways enjoined when studying tumors to first deter- 
mine their " genesis." The purpose of this direction 
(which was really quite impracticable) was that the 
transitions of diseased into normal tissue should be 
studied, and even now examinations directed to this 
end are often advisable. Only do not imagine that 
just as soon as transitional forms have been deter- 
mined the entire history of development has been 
discovered ; in this way the most serious errors have 
been made. 



INDEX 



Abbe's apparatus, 2. 
Aberration of light, 5. 
Abscess, examination of pus from, 
156. 

tubercle-bacilli in, 158. 
Absolute alcohol, 24, 184. 
Accessory apparatus, 8. 
Acetate of potassium, 40. 
Acetic acid, 18, 30. 
Acid, acetic, 30. 

chromic, 34. 

nitric, as a decolorizer, 88. 

osmic, 89. 
Acids, use of mineral, in decalcifica- 
tion, 28. 
Actinomycetes, 158. 
Albumin, coagulation of, by boiling, 

93. 
Albuminates, coagulation of, by alco- 
hol, 25. 
Alcohol, effect of, on specimens, 24. 
Alkalies, 36. 
Alum-carmine, 56. 
Alum-cochineal solution, 57. 
Amoeboid movements, 116. 
Ammonia-carmine, 51. 
Ammonium sulphide, 91. 
Amyloid substance, 48. 
Anaemia, blood-corpuscles in, 128. 

mountaineers', 177. 
Anchylomum duodenale, 5. 
Angle of divergence, 177. 
Anguillula intestinalis, 177. 
Aniline-black, 62. 

-blue, 62. 

-dyes, 67. 
Anthrax, bacillus of, 132. 
Aqueous humor, 24. 
Argand burner, 8. 
Artificial digestion, 94. 



Artificial gastric juice, 95. 

products, 19. 

serum, 24. 
Asthma-crystals, 136. 
Axis-cylinders of nerves, staining of, 
52. 

Bacilli, identification of, 68. 

of anthrax, 132. 

intestinal, 178. 

of leprosy, 75. 

of recurrent fever, 132. 

of typhus, 69, 74. 
Bacilli of tubercle, 132, 147, 166, 179. 

importance of, 151, 153. 

identification when unstained, 69. 

staining of, 78. 
Bacteria, in urine, 165. 
Base, Scjireiner's, 175. 
Basic aniline dyes, 62. 
Berlin-blue, 100, 101. 
Bismarck-brown, 64, 77. 
Blood, examination of, 126, 131. 

cellular elements in, 126. 

red corpuscles of, 128. 

white corpuscles of, 129. 

change in protoplasm of, 130. 

crystals of, 135, 136. 

parasites in, 131. 
anthrax-bacilli in, 132. 
spirilli in, 132, 133. 
Blood-plates of Bizzozero, 127. 
Blood-stains, examination of, 134. 
Boiling of specimens, 93. 
Borax-carmine, 56. 
Buhl, theory of phthisis, 144. 

Canada balsam, 40. 
Capillaries, micrococci in, 70. 
Carcinoma, cell-columns in, 171. 
diagnosis of, 170. 



192 



INDEX. 



Carcinoma of uterus, 169. 
Carmine, 5J., 53. 

alum-c, 56. 

ammonia-c., 51. 

borax-c, 56. 

fluid-injection of, 102. 

lithium-c, 57. 
Carmine-cement, 102. 
Casts, 161. 

after fractures, 163. 

epithelial, 162. 

granular, 162. 

hyaline, 161. 

waxy, 162. 
Catarrh, oral, cells in, 138. 
Cedar, oil of, 6. 
Celloidin, 99. 
Cells, amoeboid movements of, 116. 

decidual, 168. 

of endometrium, 168. 

endothelial, in blood, 131. 
Cells, epithelial, in carcinoma, 170. 

in oral fluids, 138. 

in sputa, 141. 

in stools, 176. 

in urine, 163. 
Cells, fatty degenerated in pus, 155. 

food-cells, 68. 
Cells, mucus-, in stools, 176. 

in urine, 163. 
Chloride of gold, 87. 
Chloroform, 27. 
Cholesterin, 51. 
Chromic acid, 34. 
Chyle, in exudations, 180. 
Circulation, observation of, 1*09. 

in the frog's web, 110. 

in the tongue, 110. 

in the mesentery, 111. 

in the cornea, 112. 
Clearing action of glycerin, 38. 

of Canada balsam, 40. 
Clostridium outyricum, 178. 
Cloves, oil of, 41. 
Coagulation-necrosis, 65. 
Cobalt-flasks for reagents, 18. 
Cobbler's globe, 9. 
Cochineal-alum solution, 57. 
Concretions, intestinal, 176. 

in tonsils, 140. 
Condenser, Abbe's, 3. 
Connective tissue, action of acetic 

acid on, 32. 
Cornea, circulation in, 112. 
Corpora amylacea, 48. 
Corpuscles, of blood, 127, 129. 

of pus, 155. 



Corpuscles, salivary, 139. 

of syphilis, 127. 
Correction-mountings, 6. 
Cover-glasses, 9. 
Crystals, asthma-, 136. 

haematoidin-, 136. 

haemin-, 135. 

haemoglobin-, 136. 

spermatic, 175. 
Curschman's method of staining 

amyloid, 68. 
Cylinder-diaphragm, 2. 
Cysts, contents of, 179. 

Decalcification, 28. 
Degeneration, amyloid, 49, 50. 

fatty, 25. 

senile, 187. 
Dehydration, with alcohol, 28. 
Deposits of lime, 36. 

urinary, 159. 
Detritus, significance of, in capilla- 
ries, 70. 
Diabetes, glycogen in, 48. 
Diagnosis, care necessary in making, 

185. 
Diaphragm, cylindrical, 2. 
Digestion, artificial, 94. 
Distilled water, 22. 
Distoma hcematobium, 132. 
Double knife, 11. 
Drawing, apparatus for, 7. 
Dried preparations, Koch's method 

of staining, 121. 
Drying of specimens, 94. 
Dyeing, technique of, 43. 
Dyes, aniline, 62, 6Q. 

carmine, 51, 53. 
Dysmenorrhoea, pseudomembranous, 
168. 

Echinococci, in exudations, 182. 
Egg-albumin in artificial serum, 24. 
Election of dyes, 43. 
Elements in tissue-fluids, 116. 
Eosin, 131. 

Eosinophil granules, 131. 
Epithelium. See Cells, epithelial. 
Epithelioma, true application of 

term, 172. 
Erlitzki's hardening fluid, 35. 
Erosion of cervix, distinguished 

from cancer, 169. 
Errors in pathological histology, 

186. 
Ether, 27. 
Entozoa in alimentary canal, 177. 



INDEX. 



193 



Extract of pancreas, 95. 
Exudations, 177. 
Eye-pieces, 7. 

Fat, in exudations, 180. 

in pus-corpuscles, 155. 

removal of, 28. 

in urine after fractures, 1G3. 
Fever, recurrent, spirilli in, 132. 

typhus, bacilli of, 74. 
Fibers, elastic, in sputa, 145. 
Fibrinous casts, 162. 
Filaria sanguinis, 132. 
Fluid, decalcifying, 29. 

examination of, 113. 

indifferent, 23, 24. 

Muller's, 34. 

suspended elements in, 114. 

tissue-,' 117. 
Formic acid, 33. 
Food-cells^, 66. 

Forms of cells, difficulty in determin- 
ing, 116. 
Freezing specimens, 14. 

Gangrene, sputum in, 145. 
Gastric juice, artificial, 95. 
Genital tract, secretions of, 166. 
Glass apparatus, 9. 
Globe, cobbler's, 9. 
Glycerin, 38, 40. 
Glycogen, 47, 181. 
Gold, chloride of, 87. 
Gonococcus, 173. 
Gonorrhoea, secretions of, 173. 
Gram's method of staining schizo- 

mycetes, 75. 
Granules, eosinophil, 131. 
Gum-arabic, 108. 

Haematoblasts, 127. 
Haematoidin, 136. 
Hematoxylin, 58. 
Haemin, 135. 
Haemoglobin, 136. 

Hardening in absolute alcohol, 24, 
184. 

in chromic acid, 34. 

in gold chloride, 88. 

in osmic acid, 89. 

in picric acid, 33. 
Hyaline casts, 161. 
Hydrochloric acid, 28. 
Hydrops adiposus, 180. 

chylosus, 180. 

Identification of schizomycetes, 68. 
of tubercle-bacilli, 85. 



Illuminating-lamp, 8. 
Imbedding, 97. 

material used for, 98. 
Immersion-lenses, 5, 6. 
Indifferent fluid, 24. 
Inflammation, microscopical obser- 
vation of, 109. 
Injecting, 100. 

apparatus for, 103. 

material used for, 100. 
Instruments, metallic, 10. 
Intestinal concretions, 176. 
Intestine, parasites in, 177. 
Iodine, 46. 

as a test for amyloid, 49. 

Juice, artificial gastric, 95. 

Leprosy, bacilli of, 75. 
Leucocytes, increased number in 
blood, 129. 

granular protoplasm of, 130. 

in exudations, 180. 

movements of, in fluids, 116. 

in sputa, 141. 
Leucocytosis, appearance of blood in, 

130. 
Light, for microscopic work, 8. 
Lime, oxalate of, in urine, 160. 

phosphate of, 161. 
Liquor potassae, 36. 

sodae, 36. 
Lithium-carmine, 57. 
Living tissues, observation of, 109. 
Lugol's solution, 46. 

Melanaemia, blood in, 131. 
Mesentery, circulation in, 111. 
Metallic instruments, 10. 
Metals, noble, 86. 
Menstrual fluid, 167. 
Micro-chemical examinations, 21. 
Micro-chemistry, 18. 
Micrococci, in blood, 133. 

in blood-vessels, 70. 

in chains, 70. 

in gonorrhceal secretion, 173. 

identification of, 69. 

of pneumonia, 154. 

in pus, 158. 

in sputa, 139. 

in urine, 165. 
Micro-organisms, examination of, 
118. 

identification of, 68. 
Microtome, 11, 16, 185. 
Mouth, fluids of, 137. 



194 



INDEX. 



Movement, amoeboid, in cells, 116. 
Mucous membrane of intestine, cells 
of, 176. 

of mouth, 138. 

of respiratory tract, 141. 

of uterus, 167. 
Miiller's fluid, 34. 

Nitric acid, 28, 83. 
Noble metals, 86. 
Nuclei, staining of, 63. 

Objectives, 4. 

Oidium albicans, 139, 166. 
Oil-immersion lenses, 6. 
Oil of cloves, 40. 

of turpentine, 103. 
Osmic acid, 89. 
Oxalate of lime in urine, 160. 
Oxaluria, 160. 

Pancreas, extract of, 95. 

Parasites, vegetable, in alimentary 

tract, 177. 
Pathological discoveries, 186. 
Phosphates in urine, 160. 
Phthisis, diabetic, 153. 

tuberculous, 150. 

diagnosis of, 151. 

prognosis of, 153. 
Picric acid, 33. 
Picro-carmine, 54. 
Picro-lithium-carmine, 57. 
Pigment in lungs, 142. 
Placenta, fragments of, 167. 
Pneumococci, 154. 
Potassium, acetate of, 21. 
Pregnancy, uterine mucous mem- 
brane in, 168. 
Preparations, dried, staining of, 121. 
Preservation of specimens, 106. 
Products, artificial, 19. 
Prostatic secretion, 174. 
Protoplasm, staining of, 130. 
Pulmonary phthisis, 148. 
Pus, actinomycetes in, 158. 

corpuscles of, 154. 

examination of, 155. 

foreign substances in, 156. 

fragments of bone in, 157. 

schizomycetes in, 158. 

Razor, use of, 10. 
Reagents, 18. 

for staining, 42. 
Red blood-corpuscles, 91, 127. 
Removal of fat, 27. 



Salt-solution, 23. 
Sarcinoz ventriculi, 177. 
Schizomycetes in blood, 131 e 

in oral fluids, 146. 

in pus, 158. 

staining of, 68. 

in urine, 165. 
Schreiner's base, 175. 
Sections, mounting of, 17. 

preservation of, 106. 

staining of, 43. 

thick and thin, 14. 
Semen, 174. 
Serum, artificial, 9. 
Silver, 86. 
Specimens, alcoholic, 23. 

boiling of, 93. 

preservation of, 106. 
Spermatic crystals, 175. 
Spermatozoa, 174. 
Spirilli in recurrent fever, 132. 
Spirochozta Obermeyeri, 132. 
Splenic fever, bacilli of, 74. 
Sputa, examination of, 136. 
Staining, double, 61. 

dried preparations, 121. 

Gram's method, 75. 

of micrococci, 71. 

of nuclei, 63. 

principles of, 42. 

reagents used in, 46. 

of tubercle bacilli, 78. 
Stand, microscopic, 2. 
Stomach, contents of, 175. 
Sulphide of ammonium, 91. 
Sulphuric acid, 28. 

Teichmann's test, 136. 
Tissue-fluid, 117. 

Tissues, living, observation of, 109. 
Tongue, circulation in, 110. 
Trichomonas vaginalis, 166. 
Tubercle, bacillus of, 78, 147, 153, 

158, 166, 179. 
Tumor-elements, in exudations, 181. 

in urine, 164. 
Tumors, examination of, 183. 
Typhus, bacillus of, 74, 147. 

Urates, 159. 
Uric acid, 159. 
Urine, casts in, 161. 

cells in, 163. 

examination of, 159. 

parasites in, 164. 

tumor-cells in, 164. 



INDEX. 



195 



Uterus, cancer of, 170. 
erosion of, 169. 
fluids from, 167. 



Vagina, secretion of, 166. 
Vegetable parasites, 177. 
Vital properties of cells, 114. 



Water, abstraction of, 25. 

distilled, 22. 
Web of frog, circulation in, 110. 
Weigert's method of staining, 59. 
White corpuscles of blood, 129. 



Yeast-fungi, 177. 



THE END. 



July, 1885. 

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A DICTIONARY OF MEDICINE, including Gen- 

eral Pathology, General Therapeutics, Hygiene, and the Diseases pe- 
culiar to Women and Children. By Various Writers. 

Edited by KICHAED QUAIN, M. D., F. E. S., 
Fellow of the Royal College of Physicians; Member of the Senate of the Uni- 
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eases of the Chest at Brompton, etc. 

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treatment. General Pathology comprehends articles on the origin, charac- 
ters, and nature of disease. 

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their modes of action, and on the methods of their use, The articles de- 
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Allbutt, T. Clifford, M. A., M. D. Greenfield, W. S., M. D. 

Barnes, Eobert, M. D. Jenner, Sir William, Bart. , K. C. B. , 
Bastian, H. Charlton, M. A., M. D. M. D. 

Binz, Carl, M. D. Lego, J. Wickham, M. D. 

Bristowe, J. Syer, M. D. Nightingale, Florence. 

Brown-Sequard,C.E.,M.D.,LL.D. Paget, Sir James, Bart. 

Brunton, T. Lauder, M. D., D. Sc. Parkes, Edmund A., M. D. 

Eayrer, Sir Joseph, K. C S. I., Pavy, F. W., M. D. 

M. D., LL. D. Playfair, W. S..M. D. 

Fox, Tilbury, M. D. Simon, John, C. B., D. C L. 

Galton, Captain Douglas, E. E. (re- Thompson, Sir Henry. 

tired). Waters, A. T. H., M. D. 

Gowers, W. E., M. D. Wells, T. Spencer. 

"Not only is the work a Dictionary of a nutshell the accumulated experience of 

Medicine in its fullest sense; but it is so the leading medical men of the day. As a 

encyclopedic in its scope that it may be volume for ready reference and careful 

considered a condensed review of the en- study, it will be found of immense value 

tire field of practical medicine. Each sub- to the general practitioner and student.'"—' 

ject is marked up to date and contains in Medical Record. 



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A Hand-Book of the Diseases of the Eye, and their Treat- 
ment. By Henry R. Swanzy, A. M., M. B., F. R. C. S. I., Surgeon 
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Adelaide Hospital, Dublin. Crown 8vo, 437 pages. With 122 Illus- 
trations, and Holmgren's Tests for Color-Blindness. $3.00. 

" The above is a handy manual, intended specially for students about to under- 
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prominence, and receive careful attention. It contains the essentials without 
redundant matter, and seems admirably suited to the end in view."— Canada 
Medical and Surgical Journal. 

" This hand-book is intended for students in ophthalmology, and is among 
the best in this specialty that we have perused. The author is a clear writer 
and practical ophthalmologist, and gives here very practical instruction in this 
important department of medical science." — Buffalo Medical and Surgical Jour- 
nal. 

Elements of Practical Medicine* By Alfred H. Carter, M. D., 

Member of the Royal College of Physicians, London ; Physician to 

the Queen's Hospital, Birmingham, etc. Third edition, revised and 

enlarged. 1 vol., 12mo, cloth, $3.00. 

" Although this work does not profess to be a complete treatise on the practice 
of medicine, it is too full to be called a compend ; it is rather an introduction to 
the more exhaustive study embodied in the larger text-books. Notwithstanding 
the condensed make-up of the book, it is quite comprehensive, including even 
cutaneous and venereal diseases. It contains much valuable information, and 
we may add that it is very readable."— New York Medical Journal. 

Osteotomy and Osteoclasis, for the Correction of Deformi- 
ties of the Lower Limbs. By Charles T. Poore, M. D., Sur- 
geon to St. Mary's Free Hospital for Children, New York. 1 vol., 
8vo, 1ST pages, 50 Illustrations, cloth, $2.50. 

11 There has been a want of a concise treatise on osteotomy, and the author 
aims in this work to supply the void. Re has succeeded fully in his object, and 
presents to the profession, for their guidance in the treatment of a very common 
class of deformities, a very valuable work. . . . The subjects and diseases treated 
are such as the surgeon is compelled to consider and examine often in his daily 
practice, and the author has drawn from his own ample experience and that of 
other eminent surgeons in the preparation of a most admirable and useful 
work."— Buffalo Medical and Surgical Journal. 

"This is an interesting and practical monograph, in which the author has 
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usually resorted to for the correction of osseous deformities of the lower 
extremities. The author's style is clear and forcible, the illustrations are well 
executed and to the point, and the typographical part is creditable to the pub- 
lishers. The work will be a useful addition to the surgeon's library."— ££. Louis 
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Practical Manual of Diseases of Women and Uterine 
Therapeutics, For Students and Practitioners. By H. Mac- 
naughton Jones, M. D., F. R. C. S. I. and E., Examiner in Obstetrics, 
Royal University of Ireland ; Fellow of the Academy of Medicine in 
Ireland: and of the Obstetrical Society of London, etc. 1 vol., 
12mo, 410 pages, with 188 Illustrations, cloth, $3.00. 

41 Within a very moderate compass, this work covers the field of gynaecology 
quite fully, but it deals most prominently with the minor details of that branch— 
what may be termed every-day gynaecology. Methods of diagnosis are presented 
with much completeness, and so also are measures of treatment, including, as is 
apt not to be the case with books of this class, medicinal treatment. For these 
reasons, as well as because the author's views will be found to be eminently 
judicious, we think that the book deserves to be widely studied by general 
practitioners and students."— The New York Medical Journal. 

" Those who desire to obtain, at a minimum cost of time and money, a better 
acquaintance than the present educational facilities of this country present to 
the aspirants for gynaecological celebrity, will find in this work of Dr. Jones, con- 
veyed in clear and plain terms, if not all that the modern infinitude of female 
diseases may seem to demand, yet perhaps sufficient to serve their more press- 
ing needs, not only in the line of positive instruction, but also in that which is 
not less useful to the ambitious neophyte— salutary admonition."— Canada 
Lancet, 

Atlas of Female Pelvic Anatomy. By D. Berry Hart, 
M. D., F. R. C. P. E., Lecturer on Midwifery and Diseases of Women, 
School of Medicine, Edinburgh, etc. With Preface by Alexander 
J. C. Skene, M. D., Professor of the Medical and Surgical Diseases 
of Women, Long Island College Hospital, Brooklyn, etc. Large 4to, 
37 Plates with 150 Figures, and 89 pages descriptive text. Cloth, 
$15.00. 

41 Within recent years much has been done to weed the topographical anatomy 
of the pelvis of numerous errors which have encumbered it. Prominent amon^ 
those who have furthered this work is the author of the 'Atlas' now before us, 
and into this, his latest labor, he has entered with all his accustomed vigor. . . . 
The 'Atlas' deserves, and will surely have, a wide circulation; and we are con- 
fident that no one will rise from its careful perusal without having obtained 
clearer, more accurate, and more intelligent views in regard to the much-vexed 
questions of female anatomy, or without having formed a very high opinion of 
the author's industry, earnestness, and ability." — Edinburgh Medical Journal. 

11 As an exposition of anatomical details, in their relation to obstetrics and 
gynaecology, it has no rival in the English language, and we may predict that it 
will, for years to come, occupy the position of a standard work of reference on 
these subjects."— Glasgow Medical Journal, 

The Curability and Treatment of Pulmonary Phthisis. By 

S. Jaccoud, Professor of Medical Pathology to the Faculty of Paris ; 
Member of the Academy of Medicine ; Physician to the Lariboisiere 
Hospital, Paris, etc. Translated and edited by Montagu Lubbock, 
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A Manual of Dermatology. By A. R. Robinson, M. B., L. R. C. P. 

and S. (Edin.), Professor of Dermatology at the New York Polyclinic ; 
Professor of Histology and Pathological Anatomy at the Woman's 
Medical College of the New York Infirmary. Revised and corrected. 
8vo, 64*7 pages, cloth, $5.00. 

"It includes so much good, original work, and so well illustrates the best 
practical teachings of the subject by our most advanced men, that I regard it as 
commanding at once a place in the very front rank of all authorities. . . . " — 
James Nevins Hyde, M. D. 

"Dr. Robinson's experience has amply qualified him for the task which he as- 
sumed, and he has given us a book which commends itself to the consideration 
of the general practitioner." — Medical Age. 

"In general appearance it is similar to Duhring's excellent book, more 
valuable, however, in that it contains much later views, and also on account of 
the excellence of the anatomical description accompanying the microscopical 
appearances of the diseases spoken of."— St Louis Medical and Surgical Journal. 

"Altogether it is an excellent work, helpful to every one who consults its 
pages for aid in the study of skin-diseases. No physician who studies it will 
regret the placing of it in his library.— Detroit Lancet 

NEARLY READY— June, 1885. 

The Use of the Microscope in Clinical and Pathological 
Examinations* By Dr. Carl Friedlaender, Docent in Patho- 
logical Anatomy at Berlin. Second edition, enlarged and improved, 
with a chromo- lithograph Plate. Translated, with the permission of 
the author, by Henry C. Coe, M. D., M. R. C. S., L. R. C. P. (London), 
Pathologist to the Woman's Hospital of the State of New York. 
8vo vol. of 200 pages. 

The Diagnosis and Treatment of Diseases of the Ear. By 
Oren D. Pomeroy, M. D., Surgeon to the Manhattan Eye and Ear 
Hospital, etc. With one hundred Illustrations. New edition, revised 
and enlarged. 8vo, cloth. 

Clinical Microscopy. By Dr. Alex. Peyer. New edition, revised 
and enlarged. With ninety Plates, comprising one hundred and five 
Illustrations. Translated by A. C. Girard, M. D., Assistant Surgeon, 
U. S. Army. 

A Text-Book of Ophthalmoscopy. By Edward G. Loring, M. D. 
Part I. — The Normal Eye, Determination of Refraction, and Diseases 
of the Media. 8vo. Profusely illustrated. 



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